Methods of treatment by administering anti-connexin proteins and mimetics

ABSTRACT

Methods and compositions for modulating the activities of connexins are provided, including, for example, for use for treatment of cardiovascular, vascular, neurological, for wounds and for other indications. These compounds and methods can be used therapeutically, for example, to reduce the severity of adverse effects associated diseases and disorders where localized disruption in direct cell-cell communication or prevention of hemichannel opening is desirable.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Stage Application under 35 U.S.C.§371 of International Application No. PCT/IB2006/001961, filed on Feb.3, 2006, which claims the benefit of priority to U.S. ProvisionalApplication No. 60/650,075, filed on Feb. 3, 2005 [2004]. Thedisclosures of both are incorporated herein by reference.

FIELD

The present disclosure relates to pharmaceuticals, including agents,compounds, compositions, formulations, and methods for modulation ofgap-junctions and hemichannels that are useful, for example, in theprevention and treatment of various diseases, disorders and conditions,including but not limited to cardiovascular, neurological, and vasculardiseases, disorders and conditions.

BACKGROUND

The following includes information that may be useful in understandingthe present inventions. It is not an admission that any of theinformation provided herein is prior art, or relevant, to the presentlydescribed or claimed inventions, or that any publication or documentthat is specifically or implicitly referenced is prior art.

Coronary heart disease is the leading cause of death in most westerncountries. Mortality rates for cardiovascular disease have been reportedto vary from 29 (in males, 24 for females) per 100,000 in Canada up to213 (males, females 154) in the Russian Federation. For other Westerncountries the rate is 31-55 (43-26). Approximately half the deathsattributable to stroke are the result of medical complications, such aspneumonia and sepsis, and half are attributable to neurologicalcomplications, such as new cerebral infarction and cerebral edema.Brott, T. and Bogousslavsky, J., New Engl. J. Med. 343: 710-722 (2000).

Strokes are the third leading cause of death in developed countries andhave a devastating impact on public health. About 700,000 new strokecases occur in America every year (American Stroke Association). About25% of stroke sufferers die as a result of the stroke or its resultingcomplications. Additionally, almost 50% of stroke victims have moderateto severe health impairments and long-term disabilities. Although theincidence of ischemic stroke has declined over the past 20 years, themean age of the population has risen, resulting in a continual increasein the absolute number of strokes. Recent projections indicate that bythe year 2050, more than 1 million strokes will occur each year in theUnited States. Strokes are generally the result of several underlyingconditions that decrease the flow of blood to the brain and causedisability or death. Approximately 85 percent of strokes are ischemic innature (blood clot or blockage of a blood vessel). Foulkes M R, et al.,Stroke; 19:547-54 (1988). An ischemic stroke can be caused by a bloodclot that forms inside the artery of the brain (thrombotic stroke), orby a clot that forms somewhere else in the body and travels to the brain(embolic stroke), with thrombotic strokes representing about 52% of allischemic strokes. Thrombotic strokes are generally a result ofatherosclerosis (where blood vessels become clogged with a buildup offatty deposits, calcium, and blood clotting factors such as fibrinogenand cholesterol).

Inflammation is a multifactorial process, and is manifest in manydiseases, disorders, and conditions which have enormous cumulativehealth consequences. Inflammatory diseases, including rheumatoidarthritis, lupus, psoriasis, multiple sclerosis and asthma remain amajor cause of mortality and morbidity worldwide. Autoimmune diseasesare also associated with inflammation and on the rise, reportedlyaffecting more than 50 million people in the U.S. In many autoimmunediseases, cell, tissue, joint and organ damage results from theuncontrolled activation of a immense array of inflammatory pathways.Rheumatoid arthritis (RA) is one such chronic inflammatory diseasecharacterized by inflammation of the joints, leading to swelling, pain,and loss of function. RA affects at least an estimated 2.5 millionpeople in the United States, and is caused by a combination of eventsincluding an initial infection or injury, an abnormal immune response,and genetic factors. Any one of at least 80 different autoimmunediseases can result when the immune system becomes unregulated andattacks healthy tissue.

Connexins, also known as gap junction proteins, are four-passtransmembrane proteins with cytoplasmic C and N termini. Six connexinscombine together to form a ohemichannel called a “connexon.”

Gap junctions are structures that provide direct cell-to-cellcommunication. The gap junction is composed of two connecting connexons,one contributed by each of the abutting cells that upon docking form afunctional gap junction.

As they are being translated by ribosomes, connexins are inserted intothe membrane of the endoplasmic reticulum. Bennett M V, Zukin R S.Electrical coupling and neuronal synchronization in the Mammalian brain.Neuron. 2004 Feb. 19; 41(4):495-511. There they gather to formhemichannels (connexons), which are carried to the cell membrane invesicles and diffuse through the membrane until they meet a hemichannelfrom the other cell, with which they can dock to form a channel. Id.Molecules on a connexin allow it to “recognize” the other connexins intheir hemichannel and those of the other cell's hemichannel, and causecorrect alignment and formation of the channel. Kandel E R, Schwartz JH, Jessell T M. Principles of Neural Science, 4th ed., pp. 178-180.McGraw-Hill, New York (2000).

Connexin proteins have a common transmembrane topology, with fouralpha-helical transmembrane domains, two extracellular loops, acytoplasmic loop, and cytoplasmic N- and C-terminal domains. Thesequences are most conserved in the transmembrane and extracellulardomains, yet many of the key functional differences between connexinsare determined by amino-acid differences in these largely conserveddomains. Each extracellular loop contains three cysteines with invariantspacing (save one isoform) that are required for channel function. Thejunctional channel is composed of two end-to-end hemichannels, each ofwhich is a hexamer of connexin subunits. In junctional channels, thecysteines in the extracellular loops form intra-monomer disulfide bondsbetween the two loops, not intermonomer or inter-hemichannel bonds. Theend-to-end homophilic binding between hemichannels is via non-covalentinteractions. Mutagenesis studies suggest that the docking regioncontains beta structures, and may resemble to some degree thebeta-barrel structure of porin channels. The two hemichannels thatcompose a junctional channel are rotationally staggered by approximately30 degrees relative to each other so that the alpha-helices of eachconnexin monomer are axially aligned with the alpha-helices of twoadjacent monomers in the apposed hemichannel.

Each connexon or hemichannel in the membrane should, under normalconditions, remain closed until it docks with a connexon of aneighboring cell. However, the inventors believe that when a cellexpressing a hemichannel is subjected to a stress (e.g. physiological,mechanical, etc.) hemichannels can open even when they are not docked.The inhibition of extracellular hemmichannel communication includes theinhibition of the flow of small molecules through an open hemichannel toand from an extracellular or periplamic space. While not intending to bebound by or limited to any mechanism, modes of action include blocking(partial or complete) of the hemichannel, triggering internalization ofthe connexon which is then removed from the membrane, inducing aconformational change in the connexin proteins to bring about closure ofthe connexon, and masking or binding to sites involved in triggeringchannel opening (such calcium binding sites).

Antisense (AS) nucleotides to connexins and uses thereof have beendescribed. See WO00/44409 to Becker et al., filed Jan. 27, 2000,“Formulations Comprising Antisense Nucleotides to Connexins.”

BRIEF SUMMARY

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Brief Summary. The inventions described andclaimed herein are not limited to, or by, the features or embodimentsidentified in this Summary, which is included for purposes ofillustration only and not restriction.

The inventions described and claimed herein relate to anti-connexincompounds, including polypeptides (e.g. mimetic peptides andpeptiditomimetics, antibodies, and antibody fragments and syntheticconstructs) and polynucleotides (e.g., antisense polynucleotides), formodulation of connexins, hemichannels, and gap junctions in selectedtissues, cells, and patients, for example, patients suffering from or atrisk for cardiovascular conditions, inflammatory conditions,neurological conditions, vascular conditions, wounds and otherconditions and disorders, and complications thereof.

Anti-connexin compounds and compositions useful for the treatment ofvarious diseases, conditions and disorders in which modulation ofconnexins, hemichannels, and gap junctions would be of benefit, forexample, cardiovascular, neurological, vascular, and other conditions ordisorders, including wound treatment, are provided. Also provided aremethods of using these compounds and compositions as well aspharmaceutical formulations, kits, and medical devices, for example.

In one aspect, compounds, compositions, and methods for treating asubject with a vascular disorder are provided. Such methods includeadministering to the subject an anti-connexin compound, for example, anantisense compound, mimetic peptide, or other anti-connexin compound,including those provided herein, capable of inhibiting the expression,formation, or activity of a connexin, hemichannel or gap junction.

In another aspect, anti-connexin compounds, compositions, and methodsfor treating a subject with an inflammatory disorder are provided. Suchmethods include administering to the subject an antisense compound,mimetic peptide, or other anti-connexin compound, including thoseprovided herein, capable of inhibiting the expression, formation, oractivity of a connexin, hemichannel, or gap junction.

In another aspect, anti-connexin compounds, compositions, and methodsfor treating a subject having a wound are provided. Such methods includeadministering to the subject an antisense compound, mimetic peptide, orother anti-connexin compound, including those provided herein, capableof inhibiting the expression, formation, action, or activity of aconnexin, hemichannel, or gap junction.

In another aspect, anti-connexin compounds, compositions, and methodsfor treating a subject in connection with a transplant or graftingprocedure are provided. Such methods include administering to thesubject an antisense compound, mimetic peptide, or other anti-connexincompound, including those provided herein, capable of inhibiting theexpression, formation, action, or activity of a connexin hemichannel.Such methods are also capable of inhibiting and/or preventing tissueedema associated with transplant and grafting procedures.

In another aspect, anti-connexin binding proteins, including mimeticspeptides, antibodies, antibody fragments, and the like, are providedthat are capable of binding or modulating the expression, formation,action, or activity of a connexin hemichannel. Binding proteins areuseful as modulators of gap junctions and hemichannels.

In certain non-limiting embodiments, the anti-connexin compoundcomprises a peptide comprising an amino acid sequence corresponding to atransmembrane region of a connexin. Such connexins include, for example,connexins 45, 43, 26, 30, 31.1, and 37. Human connexins are a preferredspecies.

In a non-limiting but preferred embodiment, an anti-connexin compoundcomprises a peptide comprising an amino acid sequence corresponding to aportion of a transmembrane region of a connexin 45. In particularnon-limiting embodiments, for example, the anti-connexin compound is apeptide having an amino acid sequence that comprises about 3 to about 30contiguous amino acids of SEQ ID NO:62, a peptide having an amino acidsequence that comprises about 5 to about 20 contiguous amino acids ofSEQ ID NO:62, a peptide having an amino acid sequence that comprisesabout 8 to about 15 contiguous amino acids of SEQ ID NO:62, or a peptidehaving an amino acid sequence that comprises about 11, 12, or 13contiguous amino acids of SEQ ID NO:62. Other non-limiting embodimentsinclude an anti-connexin compound that is a peptide having an amino acidsequence that comprises at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 20, 25, or 30 contiguous amino acids of SEQ ID NO:62. Incertain anti-connexin compounds provided herein, mimetic peptides arebased on the extracellular domains of connexin 45 corresponding to theamino acids at positions 46-75 and 199-228 of SEQ ID NO: 62. Thus,certain peptide described herein have an amino acid sequencecorresponding to the regions at positions 46-75 and 199-228 of SEQ IDNO: 62. The peptides need not have an amino acid sequence identical tothose portions of SEQ ID NO: 62, and conservative amino acid changes maybe made such that the peptides retain binding activity or functionalactivity in the assays described herein and otherwise known in the art.In other embodiments, mimetic peptides are based on peptide targetregions within the connexin protein other than the extracellular domains(e.g. portions of SEQ ID NO:62 not corresponding to positions 46-75 and199-228).

In another non-limiting but preferred embodiment, an anti-connexincompound comprises a peptide comprising an amino acid sequencecorresponding to a portion of a transmembrane region of a connexin 43.In particular non-limiting embodiments, the anti-connexin compound is apeptide having an amino acid sequence that comprises a peptide having anamino acid sequence that comprises about 3 to about 30 contiguous aminoacids of SEQ ID NO:63, about 5 to about 20 contiguous amino acids of SEQID NO:63, a peptide having an amino acid sequence that comprises about 8to about 15 contiguous amino acids of SEQ ID NO:63, or a peptide havingan amino acid sequence that comprises about 11, 12, or 13 contiguousamino acids of SEQ ID NO:63. Other non-limiting embodiments include ananti-connexin compound that is a peptide having an amino acid sequencethat comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,25, or 30 contiguous amino acids of SEQ ID NO:63. In other anti-connexincompounds, mimetic peptides are based on the extracellular domains ofconnexin 43 corresponding to the amino acids at positions 37-76 and178-208 of SEQ ID NO: 63. Thus, certain peptides described herein havean amino acid sequence corresponding to the regions at positions 37-76and 178-208 of SEQ ID NO: 63. The peptides need not have an amino acidsequence identical to those portions of SEQ ID NO: 63, and conservativeamino acid changes may be made such that the peptides retain bindingactivity or functional activity in the assays described herein andotherwise known in the art. In other embodiments, mimetic peptides arebased on peptide target regions within the connexin protein other thanthe extracellular domains (e.g. the portions of SEQ ID NO:63 notcorresponding to positions 37-76 and 178-208).

Other particular compounds, methods of making these compounds, andmethods of measuring their activity are described in greater detailherein.

Alternatively, anti-connexin compounds, including those provided andused in certain embodiments, comprise one or more antisense compounds.Suitable antisense compounds may be selected, for example, from thegroup consisting of antisense oligonucleotides, antisensepolynucleotides, deoxyribozymes, morpholino oligonucleotides, RNAimolecules, siRNA molecules, PNA molecules, DNAzymes, and 5′-end-mutatedU1 small nuclear RNAs, and analogs of the preceding. These and othercompounds may be used alone or in combination with one more mimetic orother binding peptides.

An anti-connexin compound such as an antisense compound or mimeticpeptide may be targeted, for example, towards one or more of connexins45, 43, 26, 37, 30 and/or 31.1. In certain non-limiting but preferredembodiments, an antisense compound or mimetic peptide is targetedtowards connexin 45 or connexin 43. In certain non-limiting butpreferred embodiments, an anti-connexin compound such as an antisensecompound targeted to at least about 8 nucleobases of a nucleic acidmolecule encoding a connexin having a nucleobase sequence selected fromSEQ ID NO:12-31. An antisense compound may comprise, for example, anantisense compound targeted to at least about 12 nucleobases of anucleic acid molecule encoding a connexin having a nucleobase sequenceselected from SEQ ID NO: 12-31. In certain other embodiments, anantisense compound comprises a nucleobase sequence selected from SEQ IDNO:1-11. An antisense compound may comprise, for example, an antisenseoligonucleotide of between about 15 and about 35 nucleobases in length,for example about 25-30 or about 30.

An antisense compound may comprise, for example, an antisenseoligonucleotide comprising naturally occurring nucleobases and anunmodified internucleoside linkage (e.g. at least one modifiedinternucleoside linkage). The modified internucleoside linkage maycomprise a phosphorothioate linkage. Other antisense compounds may, forexample, comprise an oligonucleotides having at least one modified sugarmoiety. Still other antisense compounds may, for example, compriseoligonucleotides having at least one modified nucleobase.

In certain embodiments, an anti-connexin compound such as an antisensecompound or mimetic peptide may be used in combination with a secondcompound useful for reducing tissue damage or promoting healing. Thesecond compound, for example, may be a growth factor or a cytokine orthe like. Suitable coadministration compounds include, for example, FGF,NGF, NT3, PDGF, TGF, VEGF, BDGF, EGF, KGF, integrins, interleukins,plasmin, and semaphorins. For human therapy, such coadministrationcompounds as those listed above are human or of human origin.

Methods of administering anti-connexin compounds to a subject, targetorgan, target tissue, or target cell are provided in which hemichannelmodulation would be of benefit.

Methods of administering anti-connexin compounds to a subject, targetorgan, target tissue, or target cell are provided. Thus, in certainnon-limiting embodiments a subject is treated by administration of ananti-connexin compound that is capable of binding to or modulating ahemichannel for one or more of the following: cardiovascular disease,coronary heart disease, heart failure, myocardial infarction,atherosclerosis, ischemic heart disease, cardioplegic or other organtransport or storage medium, reperfusion injury, respiratory ormetabolic acidosis, pulmonary edema (including exposure to toxic gasessuch as nitrogen dioxide), vascular pathophysiology whereby endothelialcells are disrupted (such as diet induced hypercholestrolemic lesions),vascular disorders (microvascular and macrovascular), stroke,cerebrovascular disease (cerebral ischemia), thromboses, vascularinjuries resulting from trauma (e.g. subcutaneous wounds, stentinsertion, restenosis, or angioplasty), vascular damage resulting fromelevated levels of glucose (diabetes), vascular diseases of theextremities, organ ischemia, optic neuropathies, inflammation, andrheumatoid arthritis (RA), sub-chronic or chronic inflammation, epilepsy(epileptic events and lesions spread following epileptic events),diabetic retinopathy, macular degeneration, and certain otherindications described in greater detail in Harrison's, Principles ofInternal Medicine 15^(th) Edition (McGraw Hill, Inc., New York),incorporated by reference herein.

In certain other non-limiting embodiments a subject is treated byadministration of an anti-connexin compound that is capable of bindingto or otherwise modulating a hemichannel for one or more of thefollowing, for example: (1) prevention or treatment of oedema in thespinal cord following or as a result of, for example, ischaemia ortrauma; (2) prevention or treatment of blood vessel wall degradation intissues following or as a result of, for example, ischaemia or trauma(e.g. in brain, optic nerve, spinal cord and heart); (3) prevention ortreatment of inflammatory arthritis and other inflammatory disorders inwhich, for example, oedema and/or inflammation are symptomatic, or inwhich, by way of example, blood vessel die back occurs as a result of,for example, persistent inflammation; (4) prevention or treatment ofsub-acute or chronic wounds, for example, wounds to the cornea of theeye in which, by way of example, prevention of blood vessel die backallows recovery from limbal ischaemia; (5) prevention or treatment ofsub-acute or chronic wounds, for example, wounds to the cornea of theeye as a means, by way of example, to trigger re-epithelialisation; (6)treatment of burns, for example, chemical burns in the eye in order, byway of example, to trigger epithelial recovery and to bring aboutrecovery from sub-acute limbal ischaemia; (7) prevention or treatment ofsub-acute or chronic skin wounds, including, for example, diabeticulcers, in which, by way of example, prevention of continued bloodvessel die back will allow recovery from tissue ischaemia; (8) treatmentof chronic wounds, including, for example, diabetic ulcers in which, byway of example, continued expression of connexin 43, for example at theleading edge, prevents re-epithelialisation; (9) prevention or treatmentof perinatal ischaemia using connexin mimetic peptides, which may, forexample, be delivered directly to ventricles of the brain or via spinalcolumn and/or spinal cord; (10) inhibition or prevention of oedemafollowing perinatal ischaemia using connexin mimetic peptides delivered,for example, directly to ventricles of the brain or via spinal columnand/or spinal cord; (11) treatment for perinatal ischaemia usingconnexin mimetic peptides delivered systemically, for example; (12)treatment for stroke or CNS ischaemia using connexin mimetic peptidesdelivered, for example, systemically and/or directly to ventricles ofthe brain or via spinal column and/or spinal cord; (13) prevention ofepileptiform activity (e.g. epilepsy) in the brain, includingepileptiform activity following ischaemia; and/or, (14) prevention oflesion spread, oedema (and rejection) with reperfusion following organtransplantation. Anti-connexin compounds capable of binding ormodulating connexins and gap junctions for the prevention and/ortreatment of such methods and indications are provided.

An anti-connexin compound may be administered at various predeterminedtimes.

In certain non-limiting embodiments, a connexin hemichannel activity,action, expression or formation is inhibited in endothelial cells.

In certain non-limiting embodiments, a connexin hemichannel activity,action, expression or formation is inhibited in epithelial cells.

In certain non-limiting embodiments, a subject may be treated for avascular disorder comprising a stroke.

In certain non-limiting embodiments, a subject may be treated for avascular disorder comprising an ischemia. Such an ischemia may be, forexample, a tissue ischemia, a myocardial ischemia, or a cerebralischemia.

In certain non-limiting embodiments, a subject treated herein is at riskof loss of neurological function by ischemia.

In certain non-limiting embodiments, a subject may be treated for avascular disorder comprising treating or ameliorating cell death ordegeneration in the central or peripheral nervous system that may becaused by an ischemia.

In certain non-limiting embodiments, a subject may treated for avascular disorder wherein an anti-connexin compound, for example, anantisense compound, mimetic peptide, or other binding peptide isadministered in connection with a vascular or coronary procedureperformed on a subject. In other non-limiting embodiments, ananti-connexin compound, for example, an antisense compound, mimeticpeptide, or other binding peptide is administered during said vascularor coronary procedure. The anti-connexin compound may also beadministered before or after a vascular or coronary procedure, or both.

In certain non-limiting embodiments, an anti-connexin compound,including an antisense compound, mimetic peptide, or other bindingpeptide is administered within about 1 hour after a vascular or coronaryprocedure is performed, or, for example, within about 2 hours after avascular or coronary procedure is performed. In other embodiments, theanti-connexin compound is administered within about 24 hours.Anti-connexin compounds may also be administered outside thesetimeframes, as desired or necessary.

In certain embodiments, an anti-connexin compound, including anantisense compound, mimetic peptide, or other binding peptide isadministered in connection with a heart procedure, such as heartsurgery, performed on a patient.

In certain other embodiments, an anti-connexin compound, including anantisense compound, mimetic peptide, or other binding peptide isadministered in connection with a medical device for performing avascular or other procedure.

In another aspect, a pharmaceutical formulation for administration to asubject is provided, the formulation comprising a pharmaceuticallyacceptable carrier and an agent capable of modulating a connexinhemichannel and subsequent connexon formation, such as, for example, byblocking or ameliorating hemichannel expression, formation, action,and/or activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows rat spinal cord segments 24 hours after placing intoorganotypic culture. In the control cord (FIG. 1A) significant swellingis observed at the cut ends (dotted lines mark the original cuts). Theconnexin 43 specific antisense ODN treated segment (FIG. 1B) showslittle swelling or oedema.

FIG. 2 shows coomassie Blue staining of resin sections of control spinalcord after 24 hours in organotypic culture. The neurons are vacuolatedand oedematous with “blebbing” evident along the membrane wherehemichannels are opening and allowing extracellular fluids to enter thecell (arrows).

FIG. 3 shows immunohistochemical labeling of connexin 43 usingantibodies which bind to the extracellular loop (location of binding ofGap7M antibodies shown in FIG. 3A) and to the cytoplasmic carboxyl tailof the protein (location of binding of Gap1A shown in FIG. 3A). Thecytoplasmic antibody labels all connexin 43 proteins, and the Gap7M canonly label exposed extracellular loops of hemichannels, as it issterically hindered from binding to docked connexons which are formingintact channels between cells. FIG. 3B shows dual labeling with the twoantibodies in spinal cord 24 hours after a crush wound. Based onobservation of the overlap in binding of the two labeled antibodies,most of the two antibodies colocalised, indicating that a significantportion of the protein present has not docked with neighboring cellsconnexons and remains as hemichannels. Connexin 43 specific antisenseODNs prevent protein translation and few hemichannels are seen intreated cords (FIG. 3C).

FIG. 4 shows Isolectin B4 labeling of a tissue slice from a spinal cordwhich had been in organotypic culture for five days. The lectin binds toboth microglial and blood vessel endothelial cells. The capillaryvessels in this connexin 43 specific antisense ODN treated segmentremain intact after five days (arrows). In control cords few vesselsremain fully intact after two days; by five days the pedominant labelingin controls is of activated macrophage phenotype glial cells.

FIG. 5 shows sections of spinal cord tissue 5 mm rostral to a crushwound. Four hours after crushing the cord, FITC tagged BSA was injectedinto the animal tail vein, and the tissue then removed and sectioned. Inthe control cord (FIG. 5A) the dye has leaked out extensively from theblood vessels, up to 5 mm from the injury site, indicating rupture ofblood capillary vessel walls and disruption of the blood brain barrier.In the connexin 43 specific antisense ODN treated cord (FIG. 5B) thereis very little dye that is not contained within capillaries indicatingvessel integrity has been retained.

FIG. 6 shows triple labeling of sheep heart tissue in the sheep heartventricle wall adjacent to ischemic infarct tissue 24 hours afterinfarction, and within the infarct. The tissue is labeled withIsolection B4 showing the blood vessel endothelial cells (upper leftpanel), with antibodies to connexin 43 (lower left panel) and withantibodies to Gap7M (upper right panel). The Gap7M antibodies recognizeconserved extracellular loop regions of the connexin proteins (notconnexin isoform specific), but do mark hemichannels (they aresterically hindered from accessing their epitope in intact channels).FIG. 6A shows a set of four images from tissue distant from theinfarcted region showing Isolection B4 label of capillaries (top left)and connexin 43 gap junctions in the intercalated discs of myocytes(lower left). There is virtually no GAP7M label of hemichannels (topright). The bottom right image is a merge of the other three. The bloodvessels remain intact and there is no sign of damage to the myocytesthemselves. In FIG. 6B a region still away from but closer to theinfarct is shown. The same three labels and merged image are shown inthis 4-part panel. Most of the vessels are still intact but the vesselwalls are disrupted in areas. In these areas hemichannel labelcolocalises with the ruptured vessel walls. The last panel of fourimages (FIG. 6C) is within the infarcted area itself. Few capillariesremain intact apparently following extensive hemichannel expression. Theconnexin 43 label is becoming dispersed and no longer contained inintercalated discs indicating that the myocytes have now become severelydamaged. In all cases, the Gap7M antibody label does not colocalise withthe connexin 43 label (as it does in the spinal cord), indicating theyare a different gap junction protein isoform, most likely connexin 45.

FIG. 7 shows Isolectin B4 (upper panel) marking capillary endothelialcells and myomesin antibody labeling (middle panel) marking M lines inthe sacromeres of the myocytes in an infarct 24 hours after ischemia Thelower panel shows the merged image, and colocalisation of the twoantibodies is observed. This region is the same as that shown in FIG.6C. The blood capillaries are completely disrupted and normal myocytesarcomeric banding pattern has been destroyed indicating muscle celldeath is occurring in parallel with vessel wall disintegration. The topimage in the panel shows the Isolectin B4, the middle image the myomesinlabeling, and the lower image is a merger of those two.

FIG. 8 shows a bar graph representing the percentage swelling comparedto total spinal cord segment areas. A control (media only) segment,segments treated with peptides VDCFLSRPTEKT (SEQ ID NO:35) andSRPTEKTIFII (SEQ ID NO:36) which were shown in the dye uptakeexperiments to block hemichannels (shown as peptides 4 and 5respectively in FIG. 8), and a segment treated with peptide QQPGCENVCYDK(SEQ ID NO:39) which did not block hemichannels (shown as peptide 8 inFIG. 8), are shown. The peptide SRPTEKTIFII (SEQ ID NO:36, peptide 5 inFIG. 8), a superior blocker based upon histological studies, has beenused at 5 different concentrations, the lowest of which was mosteffective in reducing oedema.

FIG. 9 shows connexin hemichannel labelling in near-term fetal sheepbrains from a sham control (left) and 24 h after 30 min. of cerebralischaemia (right) using an antibody recognising the extracellular loopregions of connexin proteins. In the control brain, no connexinhemichannel labelling is observed, whereas after ischaemia there isextensive upregulation of hemichannels. Hemichannel expression isespecially high on cell bodies (horizontal arrows) and in blood vesselendothelial cells (vertical arrows). The major connexin upregulatedafter ischaemia is connexin 43.

FIG. 10 shows examples of artificial CSF i.c.v. infusion or peptideinfusion starting 90 min after a 30 min episode of cerebral ischaemia innear-term fetal sheep. The infusion in these animals was continued until72 h after ischaemia. The vehicle (artificial CSF) infusion animal(left) shows delayed onset of severe, continuous seizures (statusepilepticus; an example is shown in the insert box, top), followed by aprogressive rise in cortical impedance (bottom, a measure of cellswelling) maximal at 48 h. Seizures resolved by approximately 48 h. Thepeptidomimetic infusion (right) changed seizures from continuous to alater onset of discrete, separate seizure events. There was a markedlydelayed and attenuated rise in cerebral impedance.

FIG. 11 shows dose-response curves for optic nerve (FIG. 11A) tissueswelling and (FIG. 11B) cell death in control, and with 2.5, 5 and 10 μMconcentrations of connexin 43 specific AS-ODN. Both parameters exhibit adecreasing trend with increasing concentration. At 10 μM, swelling ofthe tissue is reduced as early as 6 h post treatment, and by 69% at 24 hwhen compared to control. Cell death at both front and middle segment ofthe optic nerve is diminished with antisense treatments.

FIG. 12 shows the percentage of swelling in control and AS-ODN treatedoptic nerves (n=6 for all time points). Oedema is more prominent in thecontrol tissue and the difference is statistically significant at alltime points investigated. Asterisks indicate statistical significancebetween the two groups.

FIG. 13: (Top panel) Propidium iodide staining of dead cells in themiddle of control (A, B, C) and AS-ODN treated (D, E, F) optic nervesegments at 2, 6 and 24 hours after ischaemic induction. Little stainingis exhibited by the connexin 43 specific AS-ODN treated group whencompared to the controls at all three time points (Lower panel). Linegraph showing the number of dead cells per unit area in the medialregion of the nerve for the control and AS-ODN treated optic nerves.Cell death in the control group initially increases, peaks at six hoursand then declines (believed to reflect tissue oedema leaving fewer cellsper unit area). Only a very slight increase in cell death even after 24hours in culture was noted for AS-ODN treated tissue. Asterisks indicatestatistical significance between treatments.

FIG. 14 shows mean blood vessel segment length in control (green) andconenxin43 specific antisense treated optic nerves (blue). At all timepoints the antisense treated nerves have longer segments indicated lessvessel breakdown as a result of connexin expression, presumably in theform of hemichannels, or via a gap junction mediated bystander effect.

All colors referenced herein are represented on a grey-scale in thecorresponding black-and-white figures.

DETAILED DESCRIPTION

Practice of the present inventions may include or employ variousconventional techniques of molecular biology (including recombinanttechniques), microbiology, cell biology, biochemistry, nucleic acidchemistry, and immunology, which are within the skill of the art. Suchtechniques are explained fully in the literature, and include but arenot limited to, by way of example only, Molecular Cloning: A LaboratoryManual, second edition (Sambrook et al., 1989) and Molecular Cloning: ALaboratory Manual, third edition (Sambrook and Russel, 2001), jointlyand individually referred to herein as “Sambrook”; OligonucleotideSynthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney,ed., 1987); Handbook of Experimental Immunology (D. M. Weir & C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller & M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987, including supplements through2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); TheImmunoassay Handbook (D. Wild, ed., Stockton Press N.Y., 1994);Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996);Methods of Immunological Analysis (R. Masseyeff, W. H. Albert, and N. A.Staines, eds., Weinheim: V C H Verlags gesellschaft mbH, 1993), Harlowand Lane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, and Harlow and Lane (1999) Using Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (jointly and individually referred to herein as Harlow andLane), Beaucage et al. eds., Current Protocols in Nucleic Acid ChemistryJohn Wiley & Sons, Inc., New York, 2000); and Agrawal, ed., Protocolsfor Oligonucleotides and Analogs, Synthesis and Properties Humana PressInc., New Jersey, 1993).

DEFINITIONS

Before further describing the inventions in general and in terms ofvarious nonlimiting specific embodiments, certain terms used in thecontext of the describing the invention are set forth. Unless indicatedotherwise, the following terms have the following meanings when usedherein and in the appended claims. Those terms that are not definedbelow or elsewhere in the specification shall have their art-recognizedmeaning.

Amino acids used in compounds provided herein (e.g. peptides andproteins) can be genetically encoded amino acids, naturally occurringnon-genetically encoded amino acids, or synthetic amino acids. Both L-and D-enantiomers of any of the above can be utilized in the compounds.The following abbreviations may be used herein for the followinggenetically encoded amino acids (and residues thereof): alanine (Ala,A); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D);cyteine (Cys, C); glycine (Gly, G); glutamic acid (Glu, E); glutamine(Gln, Q); histidine (His, H); isoleucine (Ile, I); leucine (Leu, L);lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline(Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W);tyrosine (Tyr, Y); and valine (Val, V).

Certain commonly encountered amino acids that are not geneticallyencoded and that can be present in the compounds of the inventioninclude, but are not limited to, β-alanine (b-Ala) and other omega-aminoacids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid(Dpr, Z), 4-aminobutyric acid and so forth; α-aminoisobutyric acid(Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava);methylglycine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine(t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle, J);2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl));2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F));4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);beta-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine(hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab);2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe(pNH₂));N-methyl valine (MeVal); homocysteine (hCys);3-benzothiazol-2-yl-alanine (BztAla, B); and homoserine (hSer).Additional amino acid analogs contemplated include phosphoserine,phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylicacid, statine, α-methyl-alanine, para-benzoyl-phenylalanine,propargylglycine, and sarcosine. Peptides that are encompassed withinthe scope of the invention can have any of the foregoing amino acids inthe L- or D-configuration, or any other amino acid described herein orknown in the art, whether currently or in the future.

Amino acids that are substitutable for each other generally residewithin similar classes or subclasses. As known to one of skill in theart, amino acids can be placed into different classes dependingprimarily upon the chemical and physical properties of the amino acidside chain. For example, some amino acids are generally considered to behydrophilic or polar amino acids and others are considered to behydrophobic or nonpolar amino acids. Polar amino acids include aminoacids having acidic, basic or hydrophilic side chains and nonpolar aminoacids include amino acids having aromatic or hydrophobic side chains.Nonpolar amino acids may be further subdivided to include, among others,aliphatic amino acids. The definitions of the classes of amino acids asused herein are as follows:

“Nonpolar Amino Acid” refers to an amino acid having a side chain thatis uncharged at physiological pH, that is not polar and that isgenerally repelled by aqueous solution. Examples of genetically encodedhydrophobic amino acids include Ala, Ile, Leu, Met, Trp, Tyr and Val.Examples of non-genetically encoded nonpolar amino acids include t-BuA,Cha and Nle.

“Aromatic Amino Acid” refers to a nonpolar amino acid having a sidechain containing at least one ring having a conjugated π-electron system(aromatic group). The aromatic group may be further substituted withsubstituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl,nitro and amino groups, as well as others. Examples of geneticallyencoded aromatic amino acids include phenylalanine, tyrosine andtryptophan. Commonly encountered non-genetically encoded aromatic aminoacids include phenylglycine, 2-naphthylalanine, β-2-thienylalanine,3-benzothiazol-2-yl-alanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, 4-chlorophenylalanine, 2-fluorophenylalanine,3-fluorophenylalanine and 4-fluorophenylalanine.

“Aliphatic Amino Acid” refers to a nonpolar amino acid having asaturated or unsaturated straight chain, branched or cyclic hydrocarbonside chain. Examples of genetically encoded aliphatic amino acidsinclude Ala, Leu, Val and Ile. Examples of non-encoded aliphatic aminoacids include Nle.

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is charged or uncharged at physiological pH and that has abond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Polar amino acids are generallyhydrophilic, meaning that they have an amino acid having a side chainthat is attracted by aqueous solution. Examples of genetically encodedpolar amino acids include asparagine, cysteine, glutamine, lysine andserine. Examples of non-genetically encoded polar amino acids includecitrulline, homocysteine, N-acetyl lysine and methionine sulfoxide.

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Examples of genetically encoded acidic amino acids includeaspartic acid (aspartate) and glutamic acid (glutamate).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Examples of genetically encoded basic amino acidsinclude arginine, lysine and histidine. Examples of non-geneticallyencoded basic amino acids include ornithine, 2,3-diaminopropionic acid,2,4-diaminobutyric acid and homoarginine.

“Ionizable Amino Acid” refers to an amino acid that can be charged at aphysiological pH. Such ionizable amino acids include acidic and basicamino acids, for example, D-aspartic acid, D-glutamic acid, D-histidine,D-arginine, D-lysine, D-hydroxylysine, D-ornithine, L-aspartic acid,L-glutamic acid, L-histidine, L-arginine, L-lysine, L-hydroxylysine orL-ornithine.

As will be appreciated by those having skill in the art, the aboveclassifications are not absolute. Several amino acids exhibit more thanone characteristic property, and can therefore be included in more thanone category. For example, tyrosine has both a nonpolar aromatic ringand a polar hydroxyl group. Thus, tyrosine has several characteristicsthat could be described as nonpolar, aromatic and polar. However, thenonpolar ring is dominant and so tyrosine is generally considered to benonpolar. Similarly, in addition to being able to form disulfidelinkages, cysteine also has nonpolar character. Thus, while not strictlyclassified as a hydrophobic or nonpolar amino acid, in many instancescysteine can be used to confer hydrophobicity or nonpolarity to apeptide.

In some embodiments, polar amino acids contemplated by the presentinvention include, for example, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, histidine, homocysteine, lysine,hydroxylysine, ornithine, serine, threonine, and structurally relatedamino acids. In one embodiment the polar amino is an ionizable aminoacid such as arginine, aspartic acid, glutamic acid, histidine,hydroxylysine, lysine, or ornithine.

Examples of polar or nonpolar amino acid residues that can be utilizedinclude, for example, alanine, valine, leucine, methionine, isoleucine,phenylalanine, tryptophan, tyrosine and the like.

“Anti-connexin compounds” include those compounds that affect ormodulate the activity, expression or formation of a connexin, a connexinhemichannel (connexon), or a gap junction. Anti-connexin compoundsinclude without limitation antisense compounds (e.g. antisensepolynucleotides), antibodies and binding fragments thereof, and peptidesand polypeptides which include “peptidomimetic” and “mimetic” peptides.

“Antisense compounds” include different types of molecules that act toinhibit gene expression, translation, or function, including those thatact by sequence-specific targeting of mRNAs for therapeuticapplications. Antisense compounds include antisense DNA compounds andantisense RNA compounds. While antisense oligonucleotides are apreferred form of antisense compound, the present invention comprehendsother oligomeric antisense compounds, including but not limited tooligonucleotide mimetics. The antisense compounds in accordance withthis invention preferably comprise from about 8 to about 50 nucleobases(i.e. from about 8 to about 50 linked nucleosides). Particularlypreferred antisense compounds are antisense oligonucleotides, even morepreferably those comprising from about 12 to about 30 nucleobases.Antisense compounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression. They also include phosphorothioateoligodeoxynucleotides (S-ODNs).

Antisense compounds thus include, for example, the major nucleic-acidbased gene-silencing molecules such as, for example, chemically modifiedantisense oligodeoxyribonucleic acids (ODNs), ribozymes and siRNAs(Scherer, L. J. and Rossi, J. J. Nature Biotechnol. 21: 1457-1465(2003). Antisense compounds may also include antisense molecules suchas, for example, peptide nucleic acids (PNAs) (Braasch, D. A. and Corey,D. R., Biochemistry 41, 4503-4510 (2002)), morpholinophosphorodiamidates (Heasman, J., Dev. Biol., 243, 209-214 (2002),DNAzymes (Schubert, S. et al., Nucleic Acids Res. 31, 5982-5992 (2003).Chakraborti, S. and Banerjea, A. C., Mol. Ther. 7, 817-826 (2003),Santoro, S. W. and Joyce, G. F. Proc. Natl. Acad. Sci. USA 94, 4262-4266(1997), and the recently developed 5′-end-mutated U1 small nuclear RNAs(Fortes, P. et al., Proc. Natl. Acad. Sci. USA 100, 8264-8269 (2003).

The term “antisense sequences” refers to polynucleotides havingantisense compound activity and include, but are not limited to,sequences complementary or partially complementary or corresponding to,for example, to an RNA sequence. Antisense sequences thus include, forexample, include nucleic acid sequences that bind to mRNA or portionsthereof to block transcription of mRNA by ribosomes. Antisense methodsare generally well known in the art. See, for example, PCT publicationWO94/12633, and Nielsen et al., Science 254:1497 (1991);Oligonucleotides and Analogues, A Practical Approach, edited by F.Eckstein, IRL Press at Oxford University Press (1991); AntisenseResearch and Applications (1993, CRC Press. Antisense sequences tochosen targets may or may not also result in non-specific binding tonon-target sequences. Antisense sequences with minimal, no, ornondetectable non-specific binding to non-target sequences arepreferred.

As used herein, “messenger RNA” includes not only the sequenceinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotide sequences which form the 5′-untranslatedregion, the 3′-untranslated region, and the 5′ cap region, as well asribonucleotide sequences that form various secondary structures.Oligonucleotides may be formulated in accordance with this inventionwhich are targeted wholly or in part to any of these sequences.

In general, nucleic acids (including oligonucleotides) may be describedas “DNA-like” (i.e., having 2′-deoxy sugars and, generally, T ratherthan U bases) or “RNA-like” (i.e., having 2′-hydroxyl or 2′-modifiedsugars and, generally U rather than T bases). Nucleic acid helices canadopt more than one type of structure, most commonly the A- and B-forms.It is believed that, in general, oligonucleotides which have B-form-likestructure are “DNA-like” and those which have A-form-like structure are“RNA-like”.

The term “complementary” generally refers to the natural binding ofpolynucleotides by base pairing, for example under permissive salt andtemperature conditions. For example, the sequence “A-G-T” binds to thecomplementary sequence “T-C-A”. Complementarity between twosingle-stranded molecules may be “partial”, such that only some of thenucleic acids bind, or it may be “complete”, such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid molecules has significanteffects on the efficiency and strength of the hybridization betweenthem. “Hybridizable” and “complementary” are terms that are used toindicate a sufficient degree of complementarity such that binding,preferably stable binding sufficient to carry out an intended action,for example, occurs between the DNA or RNA target and theoligonucleotide. It is understood that an oligonucleotide need not be100% complementary to its target nucleic acid sequence to behybridizable, and it is also understood that the binding may betarget-specific, or may bind to other non-target molecules so long asthe non-specific binding does not significantly or undesirably thwartthe therapeutic or other objective. An oligonucleotide is used tointerfere with the normal function of the target molecule to cause aloss or diminution of activity, and it is preferred that there is asufficient degree of complementarity to avoid non-specific or unwantedbinding of the oligonucleotide to non-target sequences under conditionsin which specific binding is desired, i.e., under physiologicalconditions in the case of in vivo assays or therapeutic treatment or, inthe case of in vitro assays, under conditions in which the assays areconducted. Absolute complementarity is not required. Polynucleotidesthat have sufficient complementarity to form a duplex having a meltingtemperature of greater than 20° C., 30° C., or 40° C. underphysiological conditions, are generally preferred.

A “disorder” is any condition that would benefit from treatment with amolecule or composition of the invention, including those described orclaimed herein. This includes chronic and acute disorders or diseasesincluding those pathological conditions that predispose the mammal tothe disorder in question.

As used herein, “subject” refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as dogs, horses, cats, sheep, pigs, cows, etc. Thepreferred subject is a human.

“Targeting” an oligonucleotide to a chosen nucleic acid target can be amultistep process. The process may begin with identifying a nucleic acidsequence whose function is to be modulated. This may be, for example, acellular gene (or mRNA made from the gene) whose expression isassociated with a particular disease state. The targeting process mayalso include determination of a site or sites within the nucleic acidsequence for the oligonucleotide interaction to occur such that thedesired effect, i.e., inhibition of protein expression, modulation ofactivity, etc., will result. Once a target site or sites have beenidentified, antisense compounds (e.g., oligonucleotides) are chosenwhich are sufficiently or desirably complementary to the target, i.e.,hybridize sufficiently and with an adequate or otherwise desiredspecificity, to give a desired activity. In the present invention,targets include nucleic acid molecules encoding one or more connexins.The targeting process may also include determination of a site or sitesfor the antisense interaction to occur such that the desired effect willresult. A preferred intragenic site, for example, is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. The translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), and may also referred to as the “AUGcodon,” the “start codon” or the “AUG start codon.” A minority of geneshave a translation initiation codon having the RNA sequence 5′-GUG,5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown tofunction in vivo. Thus, the terms “translation initiation codon” and“start codon” can encompass many codon sequences, even though theinitiator amino acid in each instance is typically methionine (ineukaryotes) or formylmethionine (in prokaryotes). It is also known inthe art that eukaryotic and prokaryotic genes may have two or morealternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions.

The term “oligonucleotide” includes an oligomer or polymer of nucleotideor nucleoside monomers consisting of naturally occurring bases, sugarsand intersugar (backbone) linkages. The term “oligonucleotide” alsoincludes oligomers or polymers comprising non-naturally occurringmonomers, or portions thereof, which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of properties such as, for example, enhanced cellular uptake,increased stability in the presence of nucleases, or enhanced targetaffinity. A number of nucleotide and nucleoside modifications have beenshown to make the oligonucleotide into which they are incorporated moreresistant to nuclease digestion than the native oligodeoxynucleotide(ODN). Nuclease resistance is routinely measured by incubatingoligonucleotides with cellular extracts or isolated nuclease solutionsand measuring the extent of intact oligonucleotide remaining over time,usually by gel electrophoresis. Oligonucleotides, which have beenmodified to enhance their nuclease resistance, can survive intact for alonger time than unmodified oligonucleotides. A number of modificationshave also been shown to increase binding (affinity) of theoligonucleotide to its target. Affinity of an oligonucleotide for itstarget is routinely determined, for example, by measuring the Tm(melting temperature) of an oligonucleotide/target pair, which is thetemperature at which the oligonucleotide and target dissociate.Dissociation may be detected spectrophotometrically. The greater the Tm,the greater the affinity of the oligonucleotide has for the target. Insome cases, oligonucleotide modifications which enhance target-bindingaffinity are also able to enhance nuclease resistance.

A “polynucleotide” means a plurality of nucleotides. Thus, the terms“nucleotide sequence” or “nucleic acid” or “polynucleotide” or“oligonculeotide” or “oligodeoxynucleotide” all refer to a heteropolymerof nucleotides or the sequence of these nucleotides. These phrases alsorefer to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA) or to any DNA-like orRNA-like material.

A polynucleotide that encodes a connexin, a connexin fragment, or aconnexin variant includes a polynucleotide encoding: the mature form ofthe connexin found in nature (and naturally occurring and speciesvariants thereof); the mature form of the connexin found in nature andadditional coding sequence, for example, a leader or signal sequence ora proprotein sequence (and naturally occurring and species variantsthereof); either of the foregoing and non-coding sequences (for example,introns or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature form(s) of the polypeptide found in nature); fragments of themature form(s) of the connexin found in nature; and, as noted, variantsof the mature form(s) of the connexin found in nature. Thus,“connexin-encoding polynucleotide” and the like encompasspolynucleotides that have only a coding sequence for a desired connexin,fragment, or variant, as well as polynucleotides that includes othernucleotides such as additional coding and/or non-coding sequences.

In the context of the invention, messenger RNA includes not only theinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotides which form a region known to suchpersons as the 5′-untranslated region, the 3′-untranslated region, the5′ cap region and intron/exon junction ribonucleotides. Thus,oligonucleotides may be formulated in accordance with the presentinvention that are targeted wholly or in part to these associatedribonucleotides as well as to the informational ribonucleotides. Theoligonucleotide may therefore be specifically hybridizable with atranscription initiation site region, a translation initiation codonregion, a 5′ cap region, an intron/exon junction, coding sequences, atranslation termination codon region or sequences in the 5′- or3′-untranslated region. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine(prokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding connexin, regardless of the sequence(s) of such codons. It isalso known in the art that a translation termination codon (or “stopcodon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAGand 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively). The terms “start codon region,” “AUG region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3) from a translationinitiation codon. This region is a preferred target region. Similarly,the terms “stop codon region” and “translation termination codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation termination codon. This region is a preferredtarget region. The open reading frame (ORF) or “coding region,” which isknown in the art to refer to the region between the translationinitiation codon and the translation termination codon, is also a regionwhich may be targeted effectively. Other preferred target regionsinclude the 5′ untranslated region (5′UTR), known in the art to refer tothe portion of an mRNA in the 5′ direction from the translationinitiation codon, and thus including nucleotides between the 5′ cap siteand the translation initiation codon of an mRNA or correspondingnucleotides on the gene and the 3′ untranslated region (3′UTR), known inthe art to refer to the portion of an mRNA in the 3′ direction from thetranslation termination codon, and thus including nucleotides betweenthe translation termination codon and 3′ end of an mRNA or correspondingnucleotides on the gene. The 5′ cap of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The 5′ cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “intron.” which are excised from apre-mRNA transcript to yield one or more mature mRNA. The remaining (andtherefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,exon-exon or intron-exon junctions, may also be preferred targetregions, and are particularly useful in situations where aberrantsplicing is implicated in disease, or where an overproduction of aparticular mRNA splice product is implicated in disease. Aberrant fusionjunctions due to rearrangements or deletions are also preferred targets.Targeting particular exons in alternatively spliced mRNAs may also bepreferred. It has also been found that introns can also be effective,and therefore preferred, target regions for antisense compoundstargeted, for example, to DNA or pre-mRNA.

The terms “peptidomimetic” and “mimetic” include naturally occurring andsynthetic chemical compounds that may have substantially the samestructural and functional characteristics of protein regions which theymimic. For connexins these may mimic, for example, the extracellularloops of opposing connexins involved in connexon-connexon docking andcell-cell channel formation.

Peptide analogs with properties analogous to those of the templatepeptide may be non-peptide drugs. “Peptide mimetics” or“peptidomimetics,” which include peptide-based compounds, also includesuch non-peptide based compounds (Fauchere, J. Adv. Drug Res. 15: 29(1986); Veber and Freidinger; TINSS; 392 (1985); and Evans et al., J.Med. Chem. 30: 1229 (1987); Beeley N., Trends Biotechnol. June; 12(6):213-6 (1994); Kieber-Emmons T, et al.; Curr Opin Biotechnol. August;8(4): 435-41 (1997). Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalent orenhanced therapeutic or prophylactic effect. Generally, peptidomimeticsare structurally identical or similar to a paradigm polypeptide (i.e., apolypeptide that has a biological or pharmacological function oractivity), but can also have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of, forexample, —CH2NH—, —CH2S-, —CH2-CH2-, —CH═CH— (cis and trans), —COCH2-,—CH(OH)CH2-, and —CH2SO—. The mimetic can be either entirely composed ofnatural amino acids, or non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also comprise anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter mimetic activity. Forexample, a mimetic composition is within the scope of the invention ifit is capable of down-regulating biological actions or activities ofconnexin proteins or connexons, such as, for example, preventing thedocking of connexons to form gap-junction-mediated cell-cellcommunication, or preventing the opening connexons to expose the cellcytoplasm to the extracellular millieu. Peptidomimetics, mimeticpeptides, and connexin modulating peptides encompass those describedsuch peptidomimetics, mimetic peptides, and connexin modulating peptidesset forth herein, as well as those as may be known in the art, whethernow known or later developed.

The term “composition” is intended to encompass a product comprising oneor more ingredients.

The terms “modulator” and “modulation” of connexin activity, as usedherein in its various forms, is intended to encompass inhibition inwhole or in part of the expression or action or activity of a connexin.Such modulators include small molecules antagonists of connexin functionor expression, antisense molecules, ribozymes, triplex molecules, andRNAi polynucleotides, gene therapy methods, etc., and others.

The phrase “percent (%) identity” refers to the percentage of sequencesimilarity found in a comparison of two or more sequences. Percentidentity can be determined electronically using any suitable software,for example. Likewise, “similarity” between two sequences (or one ormore portions of either or both of them) is determined by comparing thesequence of one sequence to a second sequence.

“Pharmaceutically acceptable” compounds and other ingredients of acomposition or formulation, for example, a carrier, diluent orexcipient, are those that are suitable for administration to a recipientthereof.

In general, the term “protein” refers to any polymer of two or moreindividual amino acids (whether or not naturally occurring) linked viapeptide bonds, as occur when the carboxyl carbon atom of the carboxylicacid group bonded to the alpha-carbon of one amino acid (or amino acidresidue) becomes covalently bound to the amino nitrogen atom of theamino group bonded to the alpha-carbon of an adjacent amino acid. Thesepeptide bond linkages, and the atoms comprising them (i.e., alpha-carbonatoms, carboxyl carbon atoms (and their substituent oxygen atoms), andamino nitrogen atoms (and their substituent hydrogen atoms)) form the“polypeptide backbone” of the protein. In addition, as used herein, theterm “protein” is understood to include the terms “polypeptide” and“peptide” (which, at times, may be used interchangeably herein).Similarly, protein fragments, analogs, derivatives, and variants are maybe referred to herein as “proteins,” and shall be deemed to be a“protein” unless otherwise indicated. The term “fragment” of a proteinrefers to a polypeptide comprising fewer than all of the amino acidresidues of the protein. A “domain” of a protein is also a fragment, andcomprises the amino acid residues of the protein often required toconfer activity or function.

The term “stringent conditions” refers to conditions that permithybridization between polynucleotides. Stringent conditions can bedefined by salt concentration, the concentration of organic solvent (forexample, formamide), temperature, and other conditions well known in theart. Stringency can be increased by reducing the concentration of salt,increasing the concentration of organic solvents, (for example,formamide), or raising the hybridization temperature. For example,stringent salt concentration will ordinarily be less than about 750 mMNaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCland 50 mM trisodium citrate, and most preferably less than about 250 mMNaCl and 25 mM trisodium citrate. Low stringency hybridization can beobtained in the absence of organic solvent, for example, formamide,while high stringency hybridization can be obtained in the presence ofan organic solvent (for example, at least about 35% formamide, mostpreferably at least about 50% formamide). Stringent temperatureconditions will ordinarily include temperatures of at least about 30°C., more preferably of at least about 37° C., and most preferably of atleast about 42° C. Varying additional parameters, for example,hybridization time, the concentration of detergent, for example, sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed, andare within the skill in the art. Stringent hybridization conditions mayalso be defined by conditions in a range from about 5° C. to about 20°C. or 25° C. below the melting temperature (Tm) of the target sequenceand a probe with exact or nearly exact complementarity to the target. Asused herein, the melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomeshalf-dissociated into single strands. Methods for calculating the Tm ofnucleic acids are well known in the art (see, for example, Berger andKimmel, Methods In Enzymology, Vol. 152: Guide To Molecular CloningTechniques, San Diego (1987): Academic Press, Inc. and Sambrook et al.,Molecular Cloning (1989): A Laboratory Manual, 2nd Ed., Vols. 1-3, ColdSpring Harbor Laboratory). As indicated by standard references, a simpleestimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see forexample, Anderson and Young, “Quantitative Filter Hybridization” inNucleic Acid Hybridization (1985)). The melting temperature of a hybrid(and thus the conditions for stringent hybridization) is affected byvarious factors such as the length and nature (DNA, RNA, basecomposition) of the probe and nature of the target (DNA, RNA, basecomposition, present in solution or immobilized, and the like), and theconcentration of salts and other components (for example for example,the presence or absence of formamide, dextran sulfate, polyethyleneglycol). The effects of these factors are well known and are discussedin standard references in the art, see for example, Sambrook, supra, andAusubel, supra. Typically, stringent hybridization conditions are saltconcentrations less than about 1.0 M sodium ion, typically about 0.01 to1.0 M sodium ion at pH 7.0 to 8.3, and temperatures at least about 30°C. for short probes (for example, 10 to 50 nucleotides) and at leastabout 60° C. for long probes (for example, greater than 50 nucleotides).As noted, stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide, in which case lower temperaturesmay be employed. In the present invention, the polynucleotide may be apolynucleotide which hybridizes to the connexin mRNA under conditions ofmedium to high stringency such as 0.03M sodium chloride and 0.03M sodiumcitrate at from about 50 to about 60 degrees centigrade.

The term “therapeutically effective amount” means the amount of thesubject compound that will elicit a desired response, for example, abiological or medical response of a tissue, system, animal or human thatis sought, for example, by a researcher, veterinarian, medical doctor,or other clinician.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventive measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

The term “vector” refers to a nucleic acid molecule amplification,replication, and/or expression vehicle in the form of a plasmid, phage,viral, or other system (be it naturally occurring or synthetic) for thedelivery of nucleic acids to cells where the plasmid, phage, or virusmay be functional with bacterial, yeast, invertebrate, and/or mammalianhost cells. The vector may remain independent of host cell genomic DNAor may integrate in whole or in part with the genomic DNA. The vectorwill generally but need not contain all necessary elements so as to befunctional in any host cell it is compatible with. An “expressionvector” is a vector capable of directing the expression of an exogenouspolynucleotide, for example, a polynucleotide encoding a binding domainfusion protein, under appropriate conditions.

As described herein, the terms “homology and homologues” includepolynucleotides that may be a homologue of sequence in connexinpolynucleotide (e.g. mRNA). Such polynucleotides typically have at leastabout 70% homology, preferably at least about 80%, 90%, 95%, 97% or 99%homology with the relevant sequence, for example over a region of atleast about 15, 20, 30, 40, 50, 100 more contiguous nucleotides (of thehomologous sequence).

Homology may be calculated based on any method in the art. For examplethe UWGCG Package provides the BESTFIT program which can be used tocalculate homology (for example used on its default settings) (Devereuxet al., Nucleic Acids Research 12, p387-395 (1984)). The PILEUP andBLAST algorithms can be used to calculate homology or line up sequences(typically on their default settings), for example as described inAltschul S. F.; J Mol Evol 36: 290-300 (1993); Altschul, S. F. et al.; JMol Biol 215: 403-10 (1990). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/L). This algorithm involvesfirst identifying high scoring sequence pair by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Extensions for the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff and Henikoff Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992)) alignments (B) of 50, expectation (1) of 10, M=5,N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul Proc. Natl. Acad.Sci. USA 90: 5873-5787 (1993). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence is less than about 1,preferably less than about 0.1, more preferably less than about 0.01,and most preferably less than about 0.001.

The homologous sequence typically differs from the relevant sequence byat least (or by no more than) about 1, 2, 5, 10, 15, 20 or moremutations (which may be substitutions, deletions or insertions). Thesemutations may be measured across any of the regions mentioned above inrelation to calculating homology. The homologous sequence typicallyhybridizes selectively to the original sequence at a level significantlyabove background. Selective hybridization is typically achieved usingconditions of medium to high stringency (for example 0.03M sodiumchloride and 0.03M sodium citrate at from about 50 degrees C. to about60 degrees C.). However, such hybridization may be carried out under anysuitable conditions known in the art (see Sambrook et al., MolecularCloning: A Laboratory Manual (1989)). For example, if high stringency isrequired, suitable conditions include 0.2×SSC at 60 degrees C. If lowerstringency is required, suitable conditions include 2×SSC at 60 degreesC.

A “cell” means any living cell suitable for the desired application.Cells include eukaryotic and prokaryotic cells.

The term “gene product” refers to an RNA molecule transcribed from agene, or a polypeptide encoded by the gene or translated from the RNA.

The term “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (for example, “recombinantpolynucleotide”), to methods of using recombinant polynucleotides toproduce gene products in cells or other biological systems, or to apolypeptide (“recombinant protein”) encoded by a recombinantpolynucleotide. Thus, a “recombinant” polynucleotide is defined eitherby its method of production or its structure. In reference to its methodof production, the process refers to use of recombinant nucleic acidtechniques, for example, involving human intervention in the nucleotidesequence, typically selection or production. Alternatively, it can be apolynucleotide made by generating a sequence comprising a fusion of twoor more fragments that are not naturally contiguous to each other. Thus,for example, products made by transforming cells with any non-naturallyoccurring vector is encompassed, as are polynucleotides comprisingsequence derived using any synthetic oligonucleotide process. Similarly,a “recombinant” polypeptide is one expressed from a recombinantpolynucleotide.

A “recombinant host cell” is a cell that contains a vector, for example,a cloning vector or an expression vector, or a cell that has otherwisebeen manipulated by recombinant techniques to express a protein ofinterest.

The connexin gene family is diverse, with 20 identified members in thesequenced human genome. Different connexin gene products combine to formgap junctions with different properties, including pore conductance,size selectivity, charge selectivity, voltage gating properties, andchemical gating properties, and are expressed in different tissues, andat different times of development or during disease processes. In recentliterature, connexins are most commonly named according to theirmolecular weights, e.g. Cx26 is the connexin protein of 26 Kd. This canlead to confusion when connexin genes from different species arecompared, e.g. human Cx36 is homologous to zebrafish Cx35. As usedherein, therefore, it is to be understood that reference to a particularconnexin is a reference to all species variants thereof, even if theirmolecular weights are different. Thus, for example, a reference to“connexin 43” means not only human connexin 43 but to the analogousconnexin in each other species, no matter whether they are also 43 Kd.Similarly, reference to a non-human “connexin 43” is a reference to theconnexin 43 analog or variant in that species. Thus, for example,reference to “horse connexin 43” is a reference to the relevant analogor variant of human connexin 43 in horse even if it does not have a 43Kd molecular weight.

Compounds

Compounds described herein are useful for ameliorating, treating, orpreventing a variety of disorders and conditions, including but notlimited to cardiovascular, inflammatory, neurological, and vascularconditions, as well as wound treatment. The compounds are also useful inpharmaceutical compositions and in connection with medical devices andprocedures, including, for example, surgeries, grafting procedures, andorgan or tissue transplants. Certain preferred compounds describedherein are capable of modulating or affecting the transport of moleculesinto and out of cells (e.g. blocking or inhibiting). Thus certainanti-connexin compounds described herein modulate cellular communication(e.g. cell to cell). Certain anti-connexin compounds modulate or effecttransmission of molecules between the cell cytoplasm and the periplasmicor extracellular space. Such compounds are generally targeted tohemmichannels (connexons), because hemichannels are independentlyinvolved in the exchange of small molecules between the cell cytoplasmand an extracellular space or tissue. Thus, a compound provided hereinmay directly or indirectly reduce coupling between cells or between acell and an extracellular space or tissue, and the modulation oftransport of molecules from a cell into an extracellular space is withinthe scope of certain compounds and embodiments of the invention.

Any molecule that is capable of eliciting a desired inhibition of thepassage (e.g. transport) of molecules through a gap junction or connexinhemichannel may be used in embodiments of the invention. Compounds thatmodulate the passage of molecules through a gap junction or connexinhemichannel are also provided in particular embodiments (e.g., thosethat modulate the passage of molecules from the cytoplasm of a cell intoan extracellular space). Such compounds may modulate the passage ofmolecules through a gap junction or connexin hemichannel with or withoutgap junction uncoupling (blocking the transport of molecules through gapjunctions). Such compounds include, for example, proteins andpolypeptides, polynucleotides, and any other organic compound, and theymay, for example block the function or expression of a gap junction or ahemichannel in whole or in part. For a listing of some gap junctioninhibitors, see for example Evans, W. H. and Boitano, S. Biochem. Soc.Trans. 29: 606-612 (2001).

Peptide and Polypeptide Connexin Inhibitors

Binding proteins, including peptides, peptide mimetics, antibodies,antibody fragments, and the like, etc., are suitable modulators of gapjunctions and hemichannels and gap junction in certain embodiments.Binding proteins include, for example, monoclonal antibodies, polyclonalantibodies, antibody fragments (including, for example, Fab, F(ab′)₂ andFv fragments; single chain antibodies; single chain Fvs; and singlechain binding molecules such as those comprising, for example, a bindingdomain, hinge, CH2 and CH3 domains, all as described in WO02/056910 byLedbetter et al. published Jul. 25, 2002); recombinant antibodies andantibody fragments which are capable of binding an antigenic determinant(i.e., that portion of a molecule, generally referred to as an epitope)that makes contact with a particular antibody or other binding molecule.These binding proteins, including antibodies, antibody fragments, and soon, may be chimeric or humanized or otherwise made to be lessimmunogenic in the subject to whom they are to be administered, and maybe synthesized, produced recombinantly, or produced in expressionlibraries. Any binding molecule known in the art or later discovered isenvisioned, such as those referenced herein and/or described in greaterdetail in the art. See Harlow, E., and Lane, D., “Antibodies: ALaboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 349 (1988), incorporated by reference herein. For example, bindingproteins include not only antibodies, and the like, but also ligands,receptors, mimetic peptides, or other binding fragments or molecules(for example, produced by phage display) that bind to a target (e.g.connexin, hemichannel, or associated molecules).

Binding molecules will generally have a desired specificity, includingbut not limited to binding specificity, and desired affinity. Affinity,for example, may be a K_(a) of greater than or equal to about 10⁴ M⁻¹,greater than or equal to about 10⁶ M⁻¹, greater than or equal to about10⁷ M⁻¹, greater than or equal to about 10⁸ M⁻¹. Affinities of evengreater than about 10⁸ M⁻¹ are suitable, such as affinities equal to orgreater than about 10⁹ M⁻¹, about 10¹⁰ M⁻¹, about 10¹¹ M⁻¹, and about10¹² M⁻¹. Affinities of binding proteins according to the presentinvention can be readily determined using conventional techniques, forexample those described by Scatchard et al., 1949 Ann. N.Y. Acad. Sci.51: 660.

Peptide inhibitors (e.g., mimetic peptides) of gap junctions andhemichannels are preferred in certain embodiments provided herein. Seefor example Berthoud, V. M. et al., Am J. Physiol. Lung Cell Mol.Physiol. 279: L619-L622 (2000); Evans, W. H. and Boitano, S. Biochem.Soc. Trans. 29: 606-612, and De Vriese A. S., et al. Kidney Int. 61:177-185 (2001).

By using data obtained from hydropathy plots, it has been proposed thata connexin contains four-transmembrane-spanning regions and two shortextra-cellular loops. Paul D L. J Cell Biol 103: 123-134 (1996). Thepositioning of the first and second extracellular regions of connexinwas further characterized by the reported production of anti-peptideantibodies used for immunolocalization of the corresponding epitopes onsplit gap junctions. Goodenough D. A. J Cell Biol 107: 1817-1824 (1988);Meyer R. A., J Cell Biol 119: 179-189 (1992).

The extracellular domains of a hemichannel contributed by two adjacentcells “dock” with each other to form complete gap junction channels.Reagents that interfere with the interactions of these extracellulardomains will impair cell-to-cell communication. Short peptidescorresponding to sequences within the extracellular loops of connexinswere reported as inhibiters of intercellular communication. Boitano S.and Evans W. Am J Physiol Lung Cell Mol Physiol 279: L623-L630 (2000).The use of peptides as inhibitors of cell-cell channel formationproduced by connexin (Cx) 32 expressed in paired Xenopus oocytes hasbeen reported. Dahl G, et al., Biophys J 67: 1816-1822 (1994). Berthoud,V. M. and Seul, K. H., summarized some of these results. Am J. Physiol.Lung Cell Mol. Physiol. 279: L619-L622 (2000). The modulation ofhemichannel function by phosphorylation of a tyrosine residue has beenreported by Jensen et al. (US2004/0092429), the teachings of which donot encompass the anti-connexin compounds and methods provided herein.

In another aspect, a anti-connexin compound comprises a peptidecomprising an amino acid sequence corresponding to a transmembraneregion (e.g. 1^(st) to 4^(th)) of a connexin (e.g. connexin 45, 43, 26,30, 31.1, and 37). In certain embodiments, the anti-connexin compoundcomprises a peptide comprising an amino acid sequence corresponding to aportion of a transmembrane region of a connexin 45. In certainembodiments, the anti-connexin compound is a peptide having an aminoacid sequence that comprises 5 to 20 contiguous amino acids of SEQ IDNO:62, a peptide having an amino acid sequence that comprises 8 to 15contiguous amino acids of SEQ ID NO:62, or a peptide having an aminoacid sequence that comprises 11 to 13 contiguous amino acids of SEQ IDNO:62. Other embodiments are directed to an anti-connexin compound thatis a peptide having an amino acid sequence that comprises at least 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 contiguous amino acids ofSEQ ID NO:62. In certain anti-connexin compounds provided herein, theextracellular domains of connexin 45 corresponding to the amino acids atpositions 46-75 and 199-228 of SEQ ID NO: 62 are used to develop theparticular peptide sequences. Thus, certain peptide described hereinhave an amino acid sequence corresponding to the regions at positions46-75 and 199-228 of SEQ ID NO: 62. The peptides need not have an aminoacid sequence identical to those portions of SEQ ID NO: 62, andconservative amino acid changes may be made such that the peptidesretain binding activity or functional activity in the assays describedherein and otherwise known in the art. In other embodiments, peptidetarget region of the connexin protein other than the extracellulardomains (e.g. the portions of SEQ ID NO:62 not corresponding topositions 46-75 and 199-228).

In other embodiments, the anti-connexin compound comprises a peptidecomprising an amino acid sequence corresponding to a portion of atransmembrane region of a connexin 43. In certain embodiments, theanti-connexin compound is a peptide having an amino acid sequence thatcomprises 5 to 20 contiguous amino acids of SEQ ID NO:63, a peptidehaving an amino acid sequence that comprises 8 to 15 contiguous aminoacids of SEQ ID NO:63, or a peptide having an amino acid sequence thatcomprises 11 to 13 contiguous amino acids of SEQ ID NO:63. Otherembodiments are directed to an anti-connexin compound that is a peptidehaving an amino acid sequence that comprises at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 20, 25, or 30 contiguous amino acids of SEQ IDNO:63. In other anti-connexin compounds, the extracellular domains ofconnexin 43 corresponding to the amino acids at positions 37-76 and178-208 of SEQ ID NO: 63 are used to develop the particular peptidesequences. Thus, certain peptide described herein have an amino acidsequence corresponding to the regions at positions 37-76 and 178-208 ofSEQ ID NO: 63. The peptides need not have an amino acid sequenceidentical to those portions of SEQ ID NO: 63, and conservative aminoacid changes may be made such that the peptides retain binding activityor functional activity in the assays described herein and otherwiseknown in the art. In other embodiments, peptide target region of theconnexin protein other than the extracellular domains (e.g. the portionsof SEQ ID NO:63 not corresponding to positions 37-76 and 178-208).

In certain non-limiting embodiments, the anti-connexin peptides comprisesequences corresponding to a portion of the connexin extracellulardomains with conservative amino acid substitutions such that peptidesare functionally active anti-connexin compounds. Exemplary conservativeamino acid substitutions include for example the substitution of anonpolar amino acid with another nonpolar amino acid, the substitutionof an aromatic amino acid with another aromatic amino acid, thesubstitution of an aliphatic amino acid with another aliphatic aminoacid, the substitution of a polar amino acid with another polar aminoacid, the substitution of an acidic amino acid with another acidic aminoacid, the substitution of a basic amino acid with another basic aminoacid, and the substitution of an ionizable amino acid with anotherionizable amino acid.

Exemplary peptides targeted to connexin 43 are shown below in Table 1.M1, 2, 3 and 4 refer to the 1^(st) to 4^(th) transmembrane regions ofthe connexin 43 protein respectively. E2 refer to the first and secondextracellular loops respectively.

TABLE 1 Peptidic Inhibitors of Intercellular Communication (cx43)FEVAFLLIQWI M3 & E2 (SEQ ID NO:32) LLIQWYIGFSL E2 (SEQ ID NO:33)SLSAVYTCKRDPCPHQ E2 (SEQ ID NO:34) VDCFLSRPTEKT E2 (SEQ ID NO:35)SRPTEKTIFII E2 & M4 (SEQ ID NO:36) LGTAVESAWGDEQ M1 & E1 (SEQ ID NO:37)QSAFRCNTQQPG E1 (SEQ ID NO:38) QQPGCENVCYDK E1 (SEQ ID NO:39)VCYDKSFPISHVR E1 (SEQ ID NO:40)

Table 2 provides additional exemplary connexin peptides used ininhibiting hemichannel or gap junction function. In other embodiments,conservative amino acid changes are made to the peptides or fragmentsthereof.

TABLE 2 Additional Peptidic Inhibitors of Intercellular Communication(cx32, cx43) Connexin Location AA's and Sequence Cx32 E1  39-7AAESVWGDEIKSSFICNTLQPGCNSVCYDHFFPISHVR (SEQ ID NO: 41) Cx32 E1  41-52ESVWGDEKSSFI (SEQ ID NO: 42) Cx32 E1  52-63 ICNTLQPGCNSV (SEQ ID NO: 43)Cx32 E1  62-73 SVCYDHFFPISH (SEQ ID NO: 44) Cx32 E2 164-188RLVKCEAFPCPNTVDCFVSRPTEKT (SEQ ID NO: 45) Cx32 E2 166-177 VKCEAFPCPNTV(SEQ ID NO: 46) Cx32 E2 177-188 VDCFVSRPTEKT (SEQ ID NO: 47) Cx32 E1 63-75 VCYDHFFPISHVR (SEQ ID NO: 48) Cx32 E1  43-59 VWGDEKSSFICNTLQPGY(SEQ ID NO: 49) Cx32 E1  46-59 DEKSSFICNTLQPGY (SEQ ID NO: 50) Cx32 E2182-192 SRPTEKTVFTV (SEQ ID NO: 51) Cx32/ E2 182-188 SRPTEKT (SEQ ID NO:52) Cx43 201-207 Cx43 E1  64-76 VCYDKSFPISHVR (SEQ ID NO: 53) Cx43 E2201-211 SRPTEKTIFII (SEQ ID NO: 54) Cx32 E1  52-63 ICNTLQPGCNSV (SEQ IDNO: 55) Cx40 E2 177-192 FLDTLHVCRRSPCPHP (SEQ ID NO: 56) Cx43 E2 180-195SLSAVYTCKRDPCPHQ (SEQ ID NO: 57) Cx43 E1  64-76 VCYDKSFPISHVR (SEQ IDNO: 58) Cx43 E2 201-211 SRPTEKTIFII (SEQ ID NO: 59) Cx43 E2 188-205KRDPCHQVDCFLSRPTEK (SEQ ID NO: 60)

Table 3 provides the extracellular loops for connexin family memberswhich are used to develop peptide inhibitors described herein. Thepeptides and provided in Table 3, and fragments thereof are used aspeptide inhibitors in certain non-limiting embodiments. In othernon-limiting embodiments, peptides comprising from about 8 to about 15,of from about 11 to about 13 amino contiguous amino acids acids of thepetides in this Table are peptide inhibitors of the invention. In otherembodiments, conservative amino acid changes are made to the peptides orfragments thereof. See Boitano S. and Evans W. Am J Physiol Lung CellMol Physiol 279: L623-L630 (2000).

TABLE 3 Extracellular loops for various connexin family members E1huCx26      KEVWGDEQADFVCNTLQPGCKNVCYDHYFPISHIR (SEQ ID NO: 66) huCx30     QEVWGDEQEDFVCNTLQPGCKNVCYDHFFPVSHIR (SEQ ID NO: 69) huCx30.3     EEVWDDEQKDFVCNTKQPGCPNVCYDEFFPVSHVR (SEQ ID NO: 70) huCX31     ERVWGDEQKDFDCNTKQPGCTNVCYDNYFPISNIR (SEQ ID NO: 71) huCx31.1     ERVWSDDHKDFDCNTRQPGCSNVCFDEFFPVSHVR (SEQ ID NO: 72) huCx32     ESVWGDEKSSFICNTLQPGCNSVCYDQFFPISHVR (SEQ ID NO: 73) huCx36     ESVWGDEQSDFECNTAQPGCTNVCYDQAFPISHIR (SEQ ID NO: 74) huCx37     ESVWGDEQSDFECNTAQPGCTNVCYDQAFPISHIR (SEQ ID NO: 75) huCx40.1     RPVYQDEQERFVCNTLQPGCANVCYDVFSPVSHLR (SEQ ID NO: 76) hUCX43     ESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVR (SEQ ID NO: 77) huCx46     EDVWGDEQSDFTCNTQQPGCENVCYDRAFPISHIR (SEQ ID NO: 78) huCx46.6     EAIYSDEQAKFTCNTRQPGCDNVCYDAFAPLSHVR (SEQ ID NO: 79) huCx40     ESSWGDEQADFRCDTIQPGCQNVCTDQAFPISHIR (SEQ ID NO: 80) hUCX45    GESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVR (SEQ ID NO: 81) E2 huCx26 MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKT (SEQ ID NO: 82) huCx30 MYVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKT (SEQ ID NO: 83) buCx30.3 LYIFHRLYKDYDMPRVVACSVEPCPHTVDCYISRPTEKK (SEQ ID NO: 84) huCx31LYLLHTLWHGFNMPRLVQCANVAPCPNIVDCYIARPTEKK (SEQ ID NO: 85) hUCX31.1 LYVFHSFYPKYILPPVVKCHADPCPNIVDCFISKPSEKN (SEQ ID NO: 86) huCx32 MYVFYLLYPGYAMVRLVKCDVYPCPNTVDCFVSRPTEKT (SEQ ID NO: 87) huCx36        LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKT (SEQ ID NO: 88) huCx37        LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKT (SEQ ID NO: 89) huCx40.1  GALHYFLFGFLAPKKFPCTRPPCTGVVDCYVSRPTEKS (SEQ ID NO: 90) huCx43  LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKT (SEQ ID NO: 91) huCx46  IAGQYFLYGFELKPLYRCDRWPCPNTVDCFISRPTEKT (SEQ ID NO: 92) huCx46.6  LVGQYLLYGFEVRPFFPCSRQPCPHVVDCFVSRPTEKT (SEQ ID NO: 93) huCx40  IVGQYFIYGIFLTTLHVCRRSPCPHPVNCYVSRPTEKN (SEQ ID NO: 94) huCx45  LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKT (SEQ ID NO: 95)

Sequences of the E2 domain of different connexin isotypes are shown withamino acids homologous to peptide SEQ ID NO:35 and peptide SEQ ID NO:36shown in bold. Note that last 4 amino acids of peptide SEQ ID NO:36 arepart of the fourth membrane domain. Table 4 provides the extracellulardomain for connexin family members which are used to develop peptideinhibitors described herein. The peptides and provided in Table 4, andfragments thereof, are used as peptide inhibitors in certainnon-limiting embodiments. In other non-limiting embodiments, peptidescomprising from about 8 to about 15, of from about 11 to about 13 aminocontiguous amino acids acids of the petides in this Table are peptideinhibitors of the invention. In other embodiments, conservative aminoacid changes are made to the peptides or fragments thereof.

TABLE 4 Extracellular domains Peptide                               VDCFLSRPTEKT (SEQ ID NO: 35) Peptide                                SRPTEKTIFII (SEQ ID NO: 36) huCx43 LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFII (SEQ ID NO: 96) huCx26MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKTVFTV (SEQ ID NO: 97) huCx30 YVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKTVFTI (SEQ ID NO: 98) huCX30.3LYIFHRLYKDYDMPRVVACSVEPCPHTVDCYISRPTEKKVFTY (SEQ ID NO: 99) huCx31 LYLLHTLWHGFNMPRLVQCANVAPCPNIVDCYIARPTEKKTY (SEQ ID NO: 100) huCx31.1LYVFHSFYPKYILPPVVKCHADPCPNIVDCFISKPSEKNIFTL (SEQ ID NO: 101) hucx32MYVFYLLYPGYAMVRLVKCDVYPCPNTVDCFVSRPTEKTVFTV (SEQ ID NO: 102) huCx36       LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKTIFII (SEQ ID NO: 103) huCx37       LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKTIFII (SEQ ID NO: 104) huCx40.1 GALHYFLFGFLAPKKFPCTRPPCTGVVDCYVSRPTEKSLLML (SEQ ID NO: 105) huCx46 IAGQYFLYGFELKPLYRCDRWPCPNTVDCFISRPTEKTIFII (SEQ ID NO: 106) huCx46.6 LVGQYLLYGFEVRPFFPCSRQPCPHVVDCFVSRPTEKTVFLL (SEQ ID NO: 107) huCX40 IVGQYFIYGIFLTTLHVCRRSPCPHPVNCYVSRPTEKNVFIV (SEQ ID NO: 108) huCx45 LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLL (SEQ ID NO: 109)

Table 5 provides peptides inhibitors of connexin 40 shown with referenceto the extracellular loops (E1 and E2) of connexin 40. The bold aminoacids are directed to the transmembrane regions of connexin 40.

TABLE 5 Cx40 peptide inhibitors E1LGTAAESSWGDEQADFRCDTIQPGCQNVCTDQAFPISHIRFWVLQ (SEQ ID NO: 110)LGTAAESSWGDEQA (SEQ ID NO: 111)           DEQADFRCDTIQP (SEQ ID NO: 112)                   TIQPGCQNVCTDQ (SEQ ID NO: 113)                           VCTDQAFPISHIR (SEQ ID NO: 114)                                AFPISHIRFWVLQ (SEQ ID NO: 115) E2MEVGFIVGQYFIYGIFLTTLHVCRRSPCPHPVNCYVSRPTEKNVFIV (SEQ ID NO: 116)MEVGFIVGQYF (SEQ ID NO: 117)      IVGQYFIYGIFL (SEQ ID NO: 118)             GIFLTTLHVCRRSP (SEQ ID NO: 119)                       RRSPCPHPVNCY (SEQ ID NO: 120)                               VNCYVSRPTEKN (SEQ ID NO: 35)                                    SRPTEKNVFIV (SEQ ID NO: 36)

Table 6 provides peptides inhibitors of connexin 45 shown with referenceto the extracellular loops (E1 and E2) of connexin 45. The bold aminoacids are directed to the transmembrane regions of connexin 45

TABLE 6 Cx45 peptide inhibitors E1LTAVGGESIYYDEQSKFVCNTEQPGCENVCYDAPAPLSHVRFWVFQ (SEQ ID NO: 121)LTAVGGESIYYDEQS (SEQ ID NO: 122)            DEQSKFVCNTEQP (SEQ ID NO:123)                     TEQPGCENVCYDA (SEQ ID NO: 124)                            VCYDAFAPLSHVR (SEQ ID NO: 125)                                  APLSHVRFWVFQ (SEQ ID NO: 126) E2FEVGFLIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLL (SEQ ID NO: 127)FEVGFLIGQYF (SEQ ID NO: 128)      LIGQYFLYGFQV (SEQ ID NO: 129)             GFQVHPFYVCSRLP (SEQ ID NO: 130)                       SRLPCHPKIDCF (SEQ ID NO: 131)                               IDCFISRPTEKT (SEQ ID NO: 36)                                    SRPTEKTIFLL (SEQ ID NO: 37)

In certain embodiments it is preferred that certain peptide inhibitorsblock hemichannels without a desired blocking of gap junctions. Whilenot wishing to be bound to any particular theory or mechanism, it isalso believed that certain mimetic peptides (e.g. VCYDKSFPISHVR, SEQ IDNO: 53) block hemichannels without causing uncoupling of gap junctions(See Leybeart et al., Cell Commun Adhes 10: 251-257 (2003)). The peptideSRPTEKTIFII (SEQ ID NO: 54) may also be used, for example to blockhemichannels without uncoupling of gap junctions. The peptideSRGGEKNVFIV (SEQ ID NO: 61) may be used that as a control sequence (DeVriese et al., Kidney Internat. 61: 177-185 (2002)). Examples of peptideinhibitors for connexin 45 YVCSRLPCHP (SEQ ID NO:132), QVHPFYVCSRL (SEQID NO:133), FEVGFLIGQYFLY (SEQ ID NO:134), GQYFLYGFQVHP (SEQ ID NO:135),GFQVHPFYVCSR (SEQ ID NO:136), AVGGESIYYDEQ (SEQ ID NO:137), YDEQSKFVCNTE(SEQ ID NO:138), NTEQPGCENVCY (SEQ ID NO:139), CYDAFAPLSHVR (SEQ IDNO:140), FAPLSHVRFWVF (SEQ ID NO:141) and LIGQY SEQ ID NO:142), QVHPF(SEQ ID NO:143), YVCSR (SEQ ID NO:144), SRLPC (SEQ ID NO:145), LPCHP(SEQ ID NO:146) and GESIY (SEQ ID NO:147), YDEQSK (SEQ ID NO: 148),SKFVCN (SEQ ID NO:149), TEQPGCEN (SEQ ID NO:150), VCYDAFAP (SEQ IDNO:151), LSHVRFWVFQ (SEQ ID NO:152) The peptides may only be 3 aminoacids in length, including SRL, PCH, LCP, CHP, IYY, SKF, QPC, VCY, APL,HVR, or longer, for example: LIQYFLYGFQVHPF (SEQ ID NO:153),VHPFYCSRLPCHP (SEQ ID NO:154), VGGESIYYDEQSKFVCNTEQPG (SEQ ID NO:155),TEQPGCENVCYDAFAPLSHVRF (SEQ ID NO:156), AFAPLSHVRFWVFQ (SEQ ID NO: 157).

In certain non-limiting embodiments, peptides comprising from about 3 toabout 30, about 8 to about 15, of from about 11 to about 13 contiguousamino acids of the peptides in Formula 1A are peptide inhibitors of theinvention.

Formula 1A

X₁-X₂-X₃-X₄-X₅-Gly-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-Cys-X₂₀-X₂₁-X₂₂-X₂₃-Pro-Gly-X₂₆-X₂₇-X₂₈-X₂₉-Cys-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-Pro-X₃₇-X₃₈-X₃₉-X₄₀-X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆(Formula 1A)

wherein X₁ and X₃₇ are independently selected from a group consisting ofLeu, Ile, Met and Val; X₂ is selected from a group consisting of Thr,Asn, Ser and Ala; X₃ is selected from a group consisting of Ala, Ser,Gly and Thr; X₄, X₁₈, X₂₉, X₄₀ and X₄₄ are independently selected from agroup consisting of Val, Ile, Met and Leu; Xs is selected from a groupconsisting of Gly, Glu, Asp, Ser and Ala; X₇, X₁₃, X₂₂ and X₂₇ areindependently selected from a group consisting of Glu, Asp, Gln and Lys;X₈, X₁₅ and X₃₈ are independently selected from a group consisting ofSer, Ala, Asn and Thr; X₉ is selected from a group consisting of Ile,Val, Leu and Met; X₁₀, X₁₁ and X₃₁ are independently selected from agroup consisting of Tyr, Phe, Trp and His; X₁₂ and X₃₂ are independentlyselected from a group consisting of Asp, Glu and Asn; X₁₄ and X₂₃ areindependently selected from a group consisting of Gln, Glu, Arg and Lys;X₁₆ is selected from a group consisting of Lys, Arg, Glu and Gln; X₁₇and X₃₄ are independently selected from a group consisting of Phe, Tyrand Trp; X₂₀ and X₂₈ are independently selected from a group consistingof Asn, Ser, His and Asp; X₂₁ is selected from a group consisting of Thrand Ser; X₃₃ and X₃₅ are independently selected from a group consistingof Ala and Ser; X₃₉ is selected from a group consisting of His, Tyr andAsn;

X₄₁ is selected from a group consisting of Arg, Lys and Gln; X₄₂ and X₄₅are independently selected from a group consisting of Phe, Tyr, Cys, Leuand Tyr; X₄₃ is selected from a group consisting of Trp, Tyr, Arg, Lys,Cys and Phe; and X₄₆ is selected from a group consisting of Gln, His,Glu, Lys, Arg, Asn and Asp.

In certain non-limiting embodiments, peptides comprising from about 3 toabout 30, about 8 to about 15, of from about 11 to about 13 contiguousamino acids of the peptides in Formula 1B are peptide inhibitors of theinvention.

Formula 1B:

X₁-X₂-X₃-X₄-X₅-X₆-X₇-Gly-X₉-X₁₀-X₁₁-X₁₂-X₁₃-Gly-X₁₅-X₁₆-X₁₇-X₁₈-Pro-X₂₀-X₂₁-X₂₂-Cys-X₂₄-X₂₅-X₂₆-Pro-Cys-X₂₉-P-X₃₁-X₃₂-X₃₃-Cys-X₃₅-X₃₆-X₃₇-X₃₈-Pro-X₄₀-X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈(Formula 1B)

and X₂₂ are independently selected from a group consisting of Val, Ile,Met and Leu; X₄ is selected from a group consisting of Gly, Glu, Asp,Ser and Ala; X₆, X₁₂ and X₂₆ are independently selected from a groupconsisting of Leu, Ile, Met and Val; X₇, X₃₂, X₃₆, X₄₄ and X₄₈ areindependently selected from a group consisting of Ile, Val, Leu and Met;X₉, and X₁₆ are independently selected from a group consisting of Gln,Glu, Arg and Lys; X₁₀, X₁₃ and X₂₁ are independently selected from agroup consisting of Tyr, Phe, Trp and His; X₁₁, X₁₅, X₂₀ and X₃₅ areindependently selected from a group consisting of Phe, Tyr and Trp; X₁₈and X₂₉ are independently selected from a group consisting of His, Tyrand Asn; X₂₄ and X₃₇ are independently selected from a group consistingof Ser, Ala, Asn and Thr; X₂₅ and X₃₈ are independently selected from agroup consisting of Arg, Lys and Gln; X₃₁ and X₄₂ are independentlyselected from a group consisting of Lys, Arg, Glu and Gln; X₃₃ isselected from a group consisting of Asp, Glu and Asn; X₄₀ and X₄₃ areindependently selected from a group consisting of Thr and Ser; X₄₁ isselected from a group consisting of Glu, Asp, Gln and Lys; and X₄₆ andX₄₇ are independently selected from a group consisting of Val, Ile, Met,Leu and Phe.

In certain non-limiting embodiments, peptides comprising from about 3 toabout 30, from about 8 to about 15, of from about 11 to about 13contiguous amino acids of the peptides in Formula 2A are peptideinhibitors of the invention.

Formula 2A

X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-Gly-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-Cys-X₁₉-X₂₀-X₂₁-X₂₂-Pro-Gly-Cys-X₂₆-X₂₇-X₂₈-Cys-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-Pro-X₃₆-X₃₇-X₃₈-X₃₉-X₄₀-X₄₁-X₄₂-X₄₃-X₄₄-X₄₅(Formula 2A)

wherein X₁ and X₄₄ are independently selected from a group consisting ofLeu, Ile, Met, Val and Pro; X₂ is selected from a group consisting ofGly, Glu, Asp, Ser and Ala; X₃ is selected from a group consisting ofThr, Ser, Asn and Ala; X₄ is selected from a group consisting of Ala,Ser, Gly and Thr; X₅, X₂₈, X₃₉ and X₄₃ are independently selected from agroup consisting of Val, Ile, Met and Leu; X₆, X₁₂, and X₂₆ areindependently selected from a group consisting of Glu, Asp, Gln and Lys;X₇, X₁₄, X₃₃ and X₃₇ are independently selected from a group consistingof Ser, Ala, Asn and Thr; X₈ and X₁₅ are independently selected from agroup consisting of Ala and Ser; X₉ is selected from a group consistingof Trp, Tyr and Phe; X₁₁ and X₃₁ are independently selected from a groupconsisting of Asp, Glu and Asn; X₁₃, X₂₁, and X₂₂ are independentlyselected from a group consisting of Gln, Glu, Arg and Lys; X₁₆ and X₃₄are independently selected from a group consisting of Phe, Tyr and Trp;X₁₇ and X₄₀ are independently selected from a group consisting of Arg,Lys and Gln; X₁₉ and X₂₇ are independently selected from a groupconsisting of Asn, Ser, His and Asp; X₂₀ is selected from a groupconsisting of Thr and Ser; X₃₀ is selected from a group consisting ofTyr, Phe, Trp and His; X₃₂ is selected from a group consisting of Lys,Arg, Glu and Gln; X₃₆ is selected from a group consisting of Ile, Val,Leu and Met; X₃₈ is selected from a group consisting of His, Tyr andAsn; X₄₁ is selected from a group consisting of Phe, Tyr, Cys, Leu andTrp; X₄₂ is selected from a group consisting of Trp, Tyr, Phe, Arg, Lysand Cys; and X₄₅ is selected from a group consisting of Gln, His, Glu,Lys, Arg, Asn and Asp.

In certain non-limiting embodiments, peptides comprising from about 3 toabout 30, from about 8 to about 15, of from about 11 to about 13contiguous amino acids of the peptides in Formula 2B are peptideinhibitors of the invention.

Formula 2B

X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-Gly-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-Cys-X₂₄-X₂₅-X₂₆-Pro-Cys-Pro-X₃₀-X₃₁-X₃₂-X₃₃-Cys-X₃₅-X₃₆-X₃₇-X₃₈-Pro-X₄₀-X₄₁-X₄₂-X₄₃-X₄₄-X₄₅-X₄₆-X₄₇-X₄₈(Formula 2B)

wherein X₁, X₅ and X₄₅ are independently selected from a groupconsisting of Phe, Tyr, Cys, Trp and Leu; X₂ is selected from a groupconsisting of Glu, Asp, Gln, Gly, His, Arg, Asn and Lys; X₃, X₁₇ and X₂₂are independently selected from a group consisting of Val, Ile, Met andLeu; X₄ is selected from a group consisting of Gly, Glu, Asp, Ser andAla; X₆, X₁₂ and X₂₆ are independently selected from a group consistingof Leu, Ile, Met and Val; X₇, X₃₂, X₃₆, X₄₄ and X₄₈ are independentlyselected from a group consisting of Ile, Val, Leu and Met; X₉, and X₁₆are independently selected from a group consisting of Gln, Glu, Arg andLys; X₁₀, X₁₃ and X₂₁ are independently selected from a group consistingof Tyr, Phe, Trp and His; X₁₁, X₁₅, X₂₀ and X₃₅ are independentlyselected from a group consisting of Phe, Tyr and Trp; X₁₈ and X₂₉ areindependently selected from a group consisting of His, Tyr and Asn; X₂₄and X₃₇ are independently selected from a group consisting of Ser, Ala,Asn and Thr; X₂₅ and X₃₈ are independently selected from a groupconsisting of Arg, Lys and Gln; X₃₁ and/or X₄₂ is selected from a groupconsisting of Lys, Arg, Glu and Gln; X₃₃ is selected from a groupconsisting of Asp, Glu and Asn; X₄₀ and X₄₃ are independently selectedfrom a group consisting of Thr and Ser; X₄₁ is selected from a groupconsisting of Glu, Asp, Gln and Lys; and X₄₆ and X₄₇ are independentlyselected from a group consisting of Val, Ile, Met, Leu and Phe.

Affinity Binding Assays for Peptides

Pull-down assays may be used as to verify protein-peptide interaction.In this assay the peptide can be tagged, with a protein-reactive orfusion tag i.e. GST (Glutathione-S-Transferase), which will be used tocapture and ‘pull-down’ a protein-binding partner via attachment to acellulose, agarose or nickel bead. Following elution of the complexutilizing either SDS-PAGE loading buffer or alternatively competitiveanalyte elution, the complex is visualised by running on an SDS-PAGE geland using Western Analysis detection methods. Art known methods ofperforming binding assays are described in Einarson, M. B. and Orlinick,J. R., “Identification of Protein-Protein Interactions with GlutathioneS-Transferase Fusion Proteins” In Protein-Protein Interactions: AMolecular Cloning Manual, Cold Spring Harbor Laboratory Press, pp. 37-57(2002), Einarson, M. B. Detection of Protein-Protein Interactions Usingthe GST Fusion Protein Pulldown Technique. In Molecular Cloning: ALaboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, pp.18.55-18.59 (2001), and Vikis, H. G. and Guan, K. L.Glutathione-S-Transferase-Fusion Based Assays for StudyingProtein-Protein Interactions. In Protein-Protein Interactions, Methodsand Applications, Methods in Molecular Biology, 261, Fu, H. Ed. HumanaPress, Totowa, N.J., pp. 175-186 (2004), each of which is incorporatedby reference in its entirety.

Interactions and affinity of proteins and peptides can be assessed usingsurface plasmon resonance technology which enables these interactions tobe measured in real time (available through BIAcore). This approach hasthe advantage of detecting low affinity protein interactions. Surfaceplasmon resonance relies on an optical phenomenon that is used tomeasure changes in the solution concentration of molecules at abiospecific surface. This signal arises in thin metal films underconditions of total internal reflection. This signal depends on therefractive index of solutions in contact with the surface. Molecules insolution exhibit changes in refractive index and thus give rise to ameasurable signal if a biospecific interaction occurs. Typically, theprotein is immobilized by one of several possible methods onto acarboxymethylated dextran-gold surface. The interacting peptide ofinterest is injected over the surface and the kinetics of binding aremeasured in real time. Art known methods of performing binding assaysare described in Schuck, P., “Reliable determination of binding affinityand kinetics using surface plasmon resonance biosensors”, CurrentOpinion in Biotechnology, 8(4):498-502 (1997), and Zhang, X., Oglesbee,M. “Use of surface plasmon resonance for the measurement of low affinitybinding interactions between HSP72 and measles virus nucleocapsidprotein.” Biological Procedures Online. 5(1): 170-181 (2003).

Functional Assays

Functional assays can be used to determine whether mimetic peptides areable to block the opening of hemichannels. HeLa human cervical cancercell line is stably transfected with Cx43, Cx45, or another particularconnexin of interest. The cells are incubated in a zero calcium solution(HBSS-HEPES containing 1 mM EGTA), which has been shown to activateconnexin hemichannels (See Braet, K., et al., “Pharmacologicalsensitivity of ATP release triggered by photoliberation ofinositol-1,4,5-trisphosphate and zero extracellular calcium in brainendothelial cells. Journal of Cellular Physiology”, 197(2): p. 205-213(2003), DeVries, S. H. and E. A. Schwartz, “Hemi-gap-junction channelsin solitary horizontal cells of the catfish retina.” Journal ofPhysiology, 445: p. 201-230 (1992), and Li, H., et al., Properties andregulation of gap junctional hemichannels in the plasma membranes ofcultured cells. Journal of Cell Biology, 134(4): p. 1019-1030 (1996)).Cells will then be incubated for 30 minutes in a solution of HBSS-HBEPEScontaining 1 mM EGTA and 2 mM propidium iodide. Propidium iodide is afluorescent dye which is membrane impermeable but because of its smallmolecular weight is able to enter through hemichannels. Propidium iodideuptake will be determined using fluorescence microscopy. Cells will beincubated with propidium iodide in the presence of the mimetic peptidesto determine if these can prevent dye uptake.

Polynucleotide and Nucleic Acid Connexin Inhibitors

Antisense compounds may also be used in certain embodiments. Antisensecompounds include polynucleotides such as antisense deoxynucleotides,morpholino nucleotides, RNAi and deoxribozymes targeted to specificconnexin isoforms which result in reduced translation of the proteinisoform and interfere with the function of cell gap junctions.Administration of these antisense compounds results in the reduction ofgap-junction-mediated cell-cell communication at the site at whichconnexin expression is down-regulated.

Antisense compounds, for example, have been used for the modulation ofthe expression for genes implicated in viral, fungal and metabolicdiseases. U.S. Pat. No. 5,166,195, proposes oligonucleotide inhibitorsof HIV. U.S. Pat. No. 5,004,810 proposes oligomers for hybridizing toherpes simplex virus Vmw65 mRNA and inhibiting replication.

Antisense compounds are provided, including oligonucleotides, for use inmodulating the function of nucleic acid molecules encoding connexins,ultimately modulating the amount of connexins produced. This isaccomplished by providing, for example, oligonucleotides whichspecifically hybridize with nucleic acids, preferably mRNA, encodingconnexins.

An antisense oligonucleotide or polynucleotide may, for example,hybridize to all or part of a connexin mRNA. Typically the antisensepolynucleotide hybridizes to the ribosome binding region or the codingregion of the connexin mRNA. The polynucleotide may be complementary toall of or a region of a connexin mRNA. For example, the polynucleotidemay be the exact complement of all or a part of connexin mRNA. Theantisense polynucleotide may inhibit transcription and/or translation ofthe connexin. Preferably the polynucleotide is a specific inhibitor oftranscription and/or translation of the connexin gene, and does notinhibit transcription and/or translation of other genes. The product maybind to the connexin gene or mRNA either (i) 5′ to the coding sequence,and/or (ii) to the coding sequence, and/or (iii) 3′ to the codingsequence. Generally the antisense polynucleotide will cause theexpression of connexin mRNA and/or protein in a cell to be reduced. Theantisense polynucleotide is generally antisense to the connexin mRNA.Such a polynucleotide may be capable of hybridizing to the connexin mRNAand may inhibit the expression of connexin by interfering with one ormore aspects of connexin mRNA metabolism including transcription, mRNAprocessing, mRNA transport from the nucleus, translation or mRNAdegradation. The antisense polynucleotide typically hybridizes to theconnexin mRNA to form a duplex which can cause direct inhibition oftranslation and/or destabilization of the mRNA. Such a duplex may besusceptible to degradation by nucleases.

Hybridization of antisense oligonucleotides with mRNA interferes withone or more of the normal functions of mRNA. The functions of mRNA to beinterfered with include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.Binding of specific protein(s) to the RNA may also be interfered with byantisense oligonucleotide hybridization to the RNA.

The overall effect of interference with mRNA function is modulation ofexpression of connexin. In the context of this invention “modulation”includes either inhibition or stimulation; i.e., either a decrease orincrease in expression. This modulation can be measured in ways whichare routine in the art, for example by Northern blot assay of mRNAexpression, or reverse transcriptase PCR, as taught in the examples ofthe instant application or by Western blot or ELISA assay of proteinexpression, or by an immunoprecipitation assay of protein expression.Effects on cell proliferation or tumor cell growth can also be measured,as taught in the examples of the instant application. Inhibition ispresently preferred.

Once the target site or sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired modulation. The antisense nucleic acids (DNA, RNA, modified,analogues, and the like) can be made using any suitable method forproducing a nucleic acid. Oligodeoxynucleotides directed to otherconnexin proteins can be selected in terms of their nucleotide sequenceby any art recognized approach, such as, for example, the computerprograms MacVector and OligoTech (from Oligos etc. Eugene, Oreg., USA).Equipment for such synthesis is available through several vendorsincluding MacVector and OligoTech (from Oligos etc. Eugene, Oreg., USA).For general methods relating to antisense polynucleotides, see AntisenseRNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988)). See also, Dagle et al., Nucleic AcidsResearch, 19: 1805 (1991). For antisense therapy, see, for example,Uhlmann et al., Chem. Reviews, 90: 543-584 (1990). Typically, at least aportion of the nucleotide sequence is known for connexins in which theinhibition of expression is desired. Polynucleotide targets may befurther identified by gene walking techniques and walking PCR. Suchapproaches for identifying unknown polynucleotide sequences adjacent toknown nucleotide sequences are well known in the art. See for exampleParker J. D., et al. Nucleic Acids Res 19:3055-60 (1991) for adescription of walking PCR). All of the preceding references, as well asothers referenced herein, are incorporated by reference herein in theirentirety. Preferably, an antisense compound is targeted to one or morespecific connexin isotypes. Particular connexin isotypes may betargeted, for example, based upon their temporal or spatial expression.Gap junctions are expressed in vascular tissues and, in certainembodiments, connexin isotypes expressed in vascular tissues (e.g.endothelial cells) are targeted. See Camelliti P. et al., Cardiovasc.Res. 62: 414-425 (2004). Endothelial cells, endothelial progenitorcells, and smooth muscle cells have been reported to express connexins37, 40, 43 and 45. See De Wit, (2004); Haefliger et al., (2004), Sohland Willecke, (2004), and Szmitko et al., (2003). It has also beenreported that connexin 43 is upregulated in the blood vessels adjacentto wound sites. Qiu C et al., Current Biology, 13: 1967-1703 (2003).

In other embodiments, other isotypes are be targeted. Specific isotypesof connexins that may be targeted by the antisense compounds include,without limitation 45, 43, 37, 31.1, 26, and others described herein. Incertain embodiments the anti-connexin compound targets connexin 43, 45and/or 40, and in others the anti-connexin compound targets connexin 7,32 and/or 26. One or more than one connexin may be targeted In certainembodiments one anti-connexin compound targets more than one connexin(e.g. connexin 43 and connexin 45). In other embodiments, more than oneanti-connexin compound is included in a formulation or composition (e.g.a pharmaceutical composition). The invention contemplated that anyconnexin may be targeted by one or more than one anti-connexin compounddescribed herein, known in the art, or later discovered.

It is preferred, but not required, that the targeted connexins arehuman. A connexin may, for example, have a nucleobase sequence selectedfrom SEQ ID NO:12-31. In certain embodiments, antisense compounds aretargeted to at least 8 nucleobases of a nucleic acid molecule encoding aconnexin having a nucleobase sequence selected from SEQ ID NO:12-31.

Connexin targets will vary depending upon the type of tissue to beengineered or remodeled and the precise sequence of the antisensepolynucleotide used in the invention will depend upon the targetconnexin protein. The connexin protein or proteins targeted by theoligonucleotides will be dependent upon the site at which downregulationis to be directed. This reflects the nonuniform make-up of gap junction(s) at different sites throughout the body in terms of connexin sub-unitcomposition. Some connexin proteins are however more ubiquitous thanothers in terms of distribution in tissue. As described herein,

Oligonucleotides either alone or in combination may be targeted towardsconnexin 45, 43, 26, 37, 30 and/or 31.1 (e.g. see SEQ. ID. NOS:12-31)which are suitable for corneal engineering or remodeling application. Inone aspect of the invention, the oligodeoxynucleotides may be unmodifiedphosphodiester oligomers. In another aspect of the invention, thepolynucleotides may be single or double stranded.

In certain non-limiting examples, antisense compounds are targeted tospecific regions of a connexin mRNA or pre-mRNA molecule, includingexons, introns, mRNA splice sites (exon-exon or intron-exon junctions),the 5′-untranslated region, and the 3′-untranslated region.

It is also contemplated that oligonucleotides targeted at separateconnexin proteins may be used in combination (for example one, two,three, four or more different connexins may be targeted). For example,ODNs targeted to connexin 45, and one or more other members of theconnexin family (such as connexin 43, 26, 30, 31.1, 37 and 43) can beused in combination. It is also contemplated that individual antisensepolynucleotides may be specific to a particular connexin, or may target1, 2, 3 or more different connexins. Specific polynucleotides willgenerally target sequences in the connexin gene or mRNA which are notconserved between connexins, whereas non-specific polynucleotides willtarget conserved sequences. Thus, in certain embodiments, antisensecompounds are targeted to at least 8 nucleobases of a nucleic acidmolecule encoding human connexin 26, connexin 30, connexin 31.1, humanconnexin 37, connexin 43, connexin 45, wherein said antisense compoundinhibits the expression of a human connexin protein in cells associatedwith the eye of said patient.

In certain embodiments, the nucleic acid molecules corresponding to aconnexin have a nucleobase sequence selected from SEQ. ID NO: 12-31included in the sequence listing of this application (corresponding toincludes for example complements, cDNA's, portions encoding a connexinprotein, etc.). In certain embodiments, the compositions target two ormore human connexin proteins and inhibit the expression of two or morehuman connexin proteins. In further certain embodiments, the antisensecompounds are antisense oligonucleotides. Exemplary antisenseoligonucleotides to connexin 43 include GTA ATT GCG GCA AGA AGA ATT GTTTCT GTC (SEQ ID NO: 1); GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (SEQ IDNO: 2); and GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT (SEQ ID NO: 3). Anexample of an antisense oligonucleotide to connexin 26 has the sequenceTCC TGA GCA ATA CCT AAC GAA CAA ATA (SEQ ID NO: 4). Exemplary antisenseoligonucleotide to connexin 37 selected include 5′ CAT CTC CTT GGT GCTCAA CC 3′ (SEQ ID NO: 5) and 5′ CTG AAG TCG ACT TGG CTT GG 3′ (SEQ IDNO: 6). Exemplary antisense oligonucleotide to connexin 30 selectedinclude 5′ CTC AGA TAG TGG CCA GAA TGC 3′ (SEQ ID NO: 7) and 5′ TTG TCCAGG TGA CTC CAA GG 3′ (SEQ ID NO: 8). Exemplary antisenseoligonucleotide to connexin 31.1 selected include 5′ CGT CCG AGC CCA GAAAGA TGA GGT C3′ (SEQ ID NO: 9); 5′ AGA GGC GCA CGT GAG ACA C3′ (SEQ IDNO: 10); and 5′ TGA AGA CAA TGA AGA TGT T 3′ (SEQ ID NO: 11).

In a further embodiment, oligodeoxynucleotides selected from thefollowing sequences are particularly suitable for down-regulatingconnexin 43 expression:

(SEQ ID NO: 1) 5′ GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC 3′ (SEQ ID NO:2) 5′ GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC 3′; and (SEQ ID NO: 3)5′ GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT 3′

In yet another embodiment, oligodeoxynucleotides selected from the groupfollowing sequences are particularly suitable for connexins 26, 37, 30,and 31.1:

(SEQ ID NO: 4) 5′ TCC TGA GCA ATA CCT AAC GAA CAA ATA 3′ (connexin26)(SEQ ID NO: 5) 5′ CAT CTC CTT GGT GCT CAA CC 3′ (connexin37) (SEQ ID NO:6) 5′ CTG AAG TCG ACT TGG CTT GG 3′ (connexin37) (SEQ ID NO: 7) 5′ CTCAGA TAG TGG CCA GAA TGC 3′ (connexin30) (SEQ ID NO: 8) 5′ TTG TCC AGGTGA CTC CAA GG 3′ (connexin30) (SEQ ID NO: 9) 5′ CGT CCG AGC CCA GAA AGATGA GGT C 3′ (connexin31.1) (SEQ ID NO: 10) 5′ AGA GGC GCA CGT GAG ACA C3′ (connexin31.1) (SEQ ID NO: 11) 5′ TGA AGA CAA TGA AGA TGT T3′ (connexin31.1)

Antisense compounds provided herein generally comprise from about 8 toabout 40 nucleobases (i.e. from about 8 to about 40 linked nucleosides),and more typically those comprising from about 12 to about 40nucleobases, and even more typically about 30 nucleobases. Antisensecompounds comprising polynucleotides may be at least about 40, forexample at least about 60 or at least about 80, nucleotides in lengthand up to 100, 200, 300, 400, 500, 1000, 2000 or 3000 or morenucleotides in length. Suitable antisense compounds include, forexample, a 30 mer ODN.

In certain embodiments, antisense compounds are targeted to at leastabout 8 nucleobases of a nucleic acid molecule encoding a connexinhaving a nucleobase sequence selected from SEQ ID NO: 12-31. In otherembodiments, the antisense compound is targeted to at least about 10, atleast about 12, at least about 14, at least about 16, at least about 18,at least about 20, at least about 25, at least about 30, and at leastabout 35 nucleobases of a nucleic acid molecule encoding a connexinhaving a nucleobase sequence selected from SEQ ID NO:12-31. The size ofthe antisense compounds, including oligonucleotides targeted to betweenat least about 8 and 35 nucleobases of a nucleic acid molecule encodinga human connexin, may be 8 nucleobases in length or longer, between 8and 100 nucleobases, between eight and 50 nucleobases, between eight and40 nucleobases, between 10 and 50 nucleobases, between 12 and 50nucleobases, between 14 and 50 nucleobases, between 16 and 50nucleobases, between 18 and 50 nucleobases, between 20 and 50nucleobases, between 25 and 50 nucleobases, between 15 and 35nucleobases in length, and the like. Other antisense compounds of theinvention may be or smaller or larger is size, for example having morethan 100 nucleobases in length.

Antisense compounds include without limitation antisenseoligonucleotides (ODN), antisense polynucleotides, deoxyribozymes,morpholino oligonucleotides, RNAi molecules or analogs thereof, siRNAmolecules or analogs thereof, PNA molecules or analogs thereof, DNAzymesor analogs thereof, 5′-end-mutated U1 small nuclear RNAs and analogsthereof.

As provided herein, the antisense compound may include the use ofoligodeoxynucleotides (ODNs). ODNs are generally about 20 nucleotides inlength and act by hybridizing to pre-mRNA and mRNA to produce asubstrate for ribonuclease H(RNase H), which specifically degrades theRNA strand of the formed RNA-DNA duplexes. If modified in a way toprevent the action of RNase H, ODNs can inhibit translation of mRNA viasteric hindrance, or inhibit splicing of pre-mRNAs. ODNs andmodifications thereof have been used to target dsDNA for the inhibitionof transcription by the formation of triple helices. ODN may be obtainedby art-recognized methods of automated synthesis and it is relativelystraightforward to obtain ODNs of any sequence and to block geneexpression via antisense base pairing.

In certain aspects, the phosphodiester backbone of ODNs can be modifiedto increase their efficacy as target-specific agents for blocking geneexpression. These backbone modifications were developed to improve thestability of the ODNs and to enhance their cellular uptake. The mostwidely used modification is one in which the nonbridging oxygen isreplaced by a sulfur atom, creating phosphorothioate ODNs. At least onephosphorothioate ODN has been approved by the FDA, and several otherphosphorothioate antisense ODNs are in earlier stages of clinical trialsfor a variety of cancers and inflammatory diseases.

The mechanisms of action of ODNs with respect to blocking gene functionvary depending upon the backbone of the ODN (Branch, A. D. Hepatology24, 1517-1529 (1996); Dias, N. and Stein, C. A. Mol. Cancer. Thor. 1,347-355 (2002); Stein, C. A. and Cohen, J. S., Cancer Res. 48, 2659-2668(1988); Zon, G. Ann. N.Y. Acad. Sci., 616, 161-172 (1990). Netnegatively charged ODNs, such as phosphodiesters and phorphorothioates,elicit RNAse H-mediated cleavage of the target mRNA. Other backbonemodifications that do not recruit RNAse H, because of their lack ofcharge or the type of helix formed with the target RNA, can beclassified as steric hindrance ODNs. Popularly used members of thislatter group include morpholinos, U-O-methyls, 2″-O-allyls, lockednucleic acids and peptide nucleic acids (PNAs). These ODNs can blocksplicing, translation, nuclear-cytoplasmic transport and translation,among other inhibition targets.

In another aspect, modulation of the connexin expression involves theuse of ribozymes. Ribozymes are RNA molecules that act as enzymes, evenin the complete absence of proteins. They have the catalytic activity ofbreaking and/or forming covalent bonds with extraordinary specificity,thereby accelerating the spontaneous rates of targeted reactions by manyorders of magnitude.

Ribozymes bind to RNA through Watson-Crick base pairing and act todegrade target RNA by catalysing the hydrolysis of the phosphodiesterbackbone. There are several different classes of ribozymes, with the‘hammerhead’ ribozyme being the most widely studied. As its nameimplies, the hammerhead ribozyme forms a unique secondary structure whenhybridized to its target mRNA. The catalytically important residueswithin the ribozyme are flanked by target-complementary sequences thatflank the target RNA cleavage site. Cleavage by a ribozyme requiresdivalent ions, such as magnesium, and is also dependent on target RNAstructure and accessibility. Co-localizing a ribozyme with a target RNAwithin the cell through the use of localization signals greatlyincreases their silencing efficiency. The hammerhead ribozymes are shortenough to be chemically synthesized or can be transcribed from vectors,allowing for the continuous production of ribozymes within cells.

The ability of RNA to serve as a catalyst was first demonstrated for theself-splicing group I intron of Tetrahymena thermophila and the RNAmoiety of RNAse. After the discovery of these two RNA enzymes,RNA-mediated catalysis has been found associated with the self-splicinggroup II introns of yeast, fungal and plant mitochondria (as well aschloroplasts) single-stranded plant viroid and virusoid RNAs, hepatitisdelta virus and a satellite RNA from Neurospora crassa mitochondria.Ribozymes occur naturally, but can also be artificially engineered forexpression and targeting of specific sequences in cis (on the samenucleic acid strand) or trans (a noncovalently linked nucleic acid). Newbiochemical activities are being developed using in vitro selectionprotocols as well as generating new ribozyme motifs that act onsubstrates other than RNA.

The group I intron of T. thermophila was the first cis-cleaving ribozymeto be converted into a trans-reacting form, which we refer to as anintron/ribozyme, making it useful both in genomic research and as apossible therapeutic. In the trans-splicing reaction, a defective exonof a targeted mRNA can be exchanged for a correct exon that iscovalently attached to the intron/ribozyme. This occurs via a splicingreaction in which the exon attached to the intron is positioned by basepairing to the target mRNA so that it can be covalently joined to the 5″end of the target transcript in a transesterification reaction. Thisreaction has been used to trans-splice wild-type sequences into sicklecell globin transcripts and mutant p53 transcripts and replace theexpanded triplets in the 3″-UTR of protein kinase transcripts in amyotonic dystrophy allele.

The endoribonuclease RNAse P is found in organisms throughout nature.This enzyme has RNA and one or more protein components depending uponthe organism from which it is isolated. The RNA component from theEscherichia coli and Bacillus subtilis enzymes can act as asite-specific cleavage agent in the absence of the protein tradercertain salt and ionic conditions. Studies of the substrate requirementsfor human and bacterial enzymes have shown that the minimal substratesfor either enzyme resemble a segment of a transfer RNA molecule. Thisstructure can be mimicked by uniquely designed antisense RNAs, whichpair to the target RNA, and serve as substrates for RNAse P-mediated,site-specific cleavage both in the test tube and in cells. It has alsobeen shown that the antisense component can be covalently joined to theRNAse P RNA, thereby directing the enzyme only to the target RNA ofinterest. Investigators have taken advantage of this property in thedesign of antisense RNAs, which pair with target mRNAs of interest tostimulate site-specific cleavage of the target and for targetedinhibition of both herpes simplex virus and cytomegalovirus in cellculture.

A number of small plant pathogenic RNAs (viroids, satellite RNAs andvirusoids), a transcript from a N. crassa mitochondrial DNA plasmid andthe animal hepatitis delta virus undergo a self-cleavage reaction invitro in the absence of protein. The reactions require neutral pH andMg²⁺. The self-cleavage reaction is an integral part of the in vivorolling circle mechanism of replication. These self-cleaving RNAs can besubdivided into groups depending on the sequence and secondary structureformed about the cleavage site. Small ribozymes have been derived from amotif found in single-stranded plant viroid and virusoid RNAs. On thebasis of a shared secondary structure and a conserved set ofnucleotides, the term “hammerhead” has been given to one group of thisself-cleavage domain. The hammerhead ribozyme is composed of 30nucleotides. The simplicity of the hammerhead catalytic domain has madeit a popular choice in the design of trans-acting ribozymes. UsingWatson-Crick base pairing, the hammerhead ribozyme can be designed tocleave any target RNA. The requirements at the cleavage site arerelatively simple, and virtually any UH sequence motif (where H is U, Cor A) can be targeted.

A second plant-derived, self-cleavage motif, initially identified in thenegative strand of the tobacco ringspot satellite RNA, has been termedthe ‘hairpin’ or “paperclip.” The hairpin ribozymes cleave RNAsubstrates in a reversible reaction that generates 2″, Y-cyclicphosphate and 5″-hydroxT1 termini-engineered versions of this catalyticmotif also cleave and turn over multiple copies of a variety of targetsin trans. Substrate requirements for the hairpin include a GUC, withcleavage occurring immediately upstream of the G. The hairpin ribozymealso catalyzes a ligation reaction, although it is more frequently usedfor cleavage reactions.

There have been numerous applications of both hammerhead and hairpinribozymes in cells for downregulating specific cellular and viraltargets. Haseloff and Gerlach designed a hammerhead motif (Haseloff andGerlach; Nature. August 18; 334(6183): 585-91 (1988)) that can beengineered to cleave any target by modifying the arms that base pairwith right target. Ramemzani et al. demonstrated that this hammerheadribozyme motif had potential therapeutic applications in a study inwhich there was a virtual complete inhibition of viral gene expressionand replication using cells engineered to express an anti-humanimmunodeficiency virus (HIV) gag ribozyme (Ramezani A. et al., Frontiersin Bioscience 7: a, 29-36; (2002)).

In another aspect, modulation of the connexin expression involves theuse of catalytic DNAs (or DNAzymes). Small DNAs capable of sitespecifically cleaving RNA targets have been developed via in vitroevolution (as no known DNA enzymes occur in nature). Two differentcatalytic motifs, with different cleavage site specificities have beenidentified. The most commonly used 10-20 enzymes bind to their RNAsubstrates via Watson-Crick base pairing and site specifically cleavethe target RNA, as do the hammerhead and hairpin ribozymes, resulting in2; 3″-cyclic phosphate and 5″-OH termini. Cleavage of the target mRNAsresults in their destruction and the DNAzymes recycle and cleavemultiple substrates. Catalytic DNAs are relatively inexpensive tosynthesize and have good catalytic properties, making them usefulsubstitutes for either antisense DNA or ribozymes.

Several applications of DNAzymes in cell culture have been publishedincluding the inhibition of veg FmRNA and consequent prevention ofangiogenesis, and inhibition of expression of the bcr/abl fusiontranscript characteristic of chronic myelogenous leukemia. CatalyticDNAs can be delivered exogenously, and they can be backbone-modified toin order to optimize systemic delivery in the absence of a carrier.

In another aspect of the present invention, the modulation of theconstitutive connexin gene involves the use of oligonucleotides havingmorpholino backbone structures. Summerton, J. E. and Weller, D. D. U.S.Pat. No. 5,034,506.

In another aspect of the invention, the antisense polynucleotides may bechemically modified in order to enhance their resistance to nucleasesand increase the efficacy of cell entry. For example, mixed backboneoligonucleotides (MBOs) containing segments of phosphothioateoligodeoxynucleotides and appropriately placed segments of modifiedoligodeoxyor oligoribonucleotides may be used. MBOs have segments ofphosphorothioate linkages and other segments of other modifiedoligonucleotides, such as methylphosphonates, phosphoramidates,phosphorodithioates, N3′P5′-phosphoramidates and oligoribonucleotidephosphorothioates and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates, which are non-ionic, andvery resistant to nucleases or 2′-O-alkyloligoribonucleotides.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

The antisense compounds useful in this invention may includeoligonucleotides containing modified backbones or non-naturalinternucleoside linkages. Oligonucleotides having modified backbonesinclude those that retain a phosphorus atom in the backbone and thosethat do not have a phosphorus atom in the backbone. In the context ofthis invention, modified oligonucleotides that do not have a phosphorusatom in their internucleoside backbone can also be considered to beoligonucleosides.

The antisense compounds with modified oligonucleotide backbones usefulin this invention may include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 51-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

In one aspect, it is contemplated that modified oligonucleotidebackbones that do not include a phosphorus atom therein have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages. These include those having morpholino linkages(formed in part from the sugar portion of a nucleoside); siloxanebackbones; sulfide, sulfoxide and sulfone backbones; formacetyl andthioformacetyl backbones; methylene formacetyl and thioformacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH2 component parts.

In one aspect, it is contemplated that oligonucleotide mimetics, boththe sugar and the internucleoside linkage, i.e. the backbone of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an oligonucleotide mimetic thathas been shown to have excellent hybridization properties, is referredto as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backboneof an oligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Further teaching of PNA compounds can be foundin Nielsen et al. (Science, 254, 1497-1500 (1991)).

In one aspect, oligonucleotides with phosphorothioate backbones andoligonucleosides with heteroatom backbones, and in particular—CH2-NH—O—CH2-, —CH-2 N(CH)3-O—CH-2 [known as a methylene (methylimino)or MMI backbone], —CH2-O—N(CH3)-CH2-, —CH2-N(CH3)-N(CH3)-CH2- and—O—N(CH3)-CH2-CH2-[wherein the native phosphodiester backbone isrepresented as —O—P—O—CH2-] are contemplated. In yet another aspect,oligonucleotides having morpholino and amide backbone structures arealso contemplated.

In another aspect, it is contemplated that the modified oligonucleotidesmay also contain one or more substituted sugar moieties. For example,oligonucleotides comprising one of the following at the 2′ position: OH;F; O-, S-, or N-alkyl, O-alkyl-O-alkyl, O-, S-, or N-alkenyl, or O-, S-or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substitutedor unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.Particularly preferred are O[(CH2)n O]m CH3, O(CH2)n OCH3, O(CH2)2ON(CH3)2, O(CH2)n NH2, O(CH2)n CH3, O(CH2)n ONH2, and O(CH2)n ON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferredoligonucleotides may comprise one of the following at the 2′ position:C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′-O—CH2 CH2 OCH, 3 also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al. Helv. Chim. Acta 1995,78, 486-504) i.e. an alkoxyalkoxy group. Other modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH2)2 ON(CH3). 2 group, also knownas 2′-DMAOE, and 2′-dimethylamino-ethoxyethoxy (2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2.

It is further contemplated that the modifications may include 2′-methoxy(2′-O—CH3), 2′-aminopropoxy (2′-OCH2 CH2 CH2 NH2) and 2′-fluoro (2′-F).Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

In another aspect, it is contemplated that the oligonucleotides may alsoinclude nucleobase (often referred to in the art simply as “base”)modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (I), cytosine (C) and uracil (U).Modified nucleobases include other synthetic and natural nucleobasessuch as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-amincadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859 (1990), Kroschwitz, J. John Wiley & Sons,those disclosed by Englisch et al. (Angewandte Chemie, InternationalEdition, 30, 613-722 (1991)), and those disclosed by Sanghvi, Y. S.,Chapter 15, Antisense Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., ed., CRC Press (1993). Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, pages 276-278 (1993)) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

In another aspect, it is contemplated that the modification of theoligonucleotides involves chemically linking to the oligonucleotide oneor more moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide. Such moietiesinclude but are not limited to lipid moieties such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 86, 6553-6556(1989)), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 4,1053-1059 (1994)), a thioether, e.g., hexyl-5-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci. 660, 306-309; Manoharan et al., Bioorg (1992).Med. Chem. Let. 3, 2765-2770 (1993)), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 20, 533-538 (1992)), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J. 10,1111-1118 (1991); Kabanov et al., FEBS Lett. 259, 327-330 (1990);Svinarchuk et al., Biochimie 75, 49-54 (1993)), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett. 36, 3651-3654 (1995); Shea et al., Nucl. Acids Res.18, 3777-3783 (1990)), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides 14, 969-973 (1995)), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett. 36,3651-3654 (1995)), a palmityl moiety (Mishra et al., Biochim. Biophys.Acta 1264, 229-237 (1995)), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther. 277, 923-937 (1996)).

Also contemplated are the use of oligonucleotides which are chimericoligonucleotides. “Chimeric” oligonucleotides or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionwherein the oligonucleotide is modified so as to confer upon theoligonucleotide increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase His a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof antisense inhibition of gene expression. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art. ThisRNAse H-mediated cleavage of the RNA target is distinct from the use ofribozymes to cleave nucleic acids.

Examples of chimeric oligonucleotides include but are not limited to“gapmers,” in which three distinct regions are present, normally with acentral region flanked by two regions which are chemically equivalent toeach other but distinct from the gap. A preferred example of a gapmer isan oligonucleotide in which a central portion (the “gap”) of theoligonucleotide serves as a substrate for RNase H and is preferablycomposed of 2′-deoxynucleotides, while the flanking portions (the 5′ and3′ “wings”) are modified to have greater affinity for the target RNAmolecule but are unable to support nuclease activity (e.g. fluoro- or2′-O-methoxyethyl-substituted). Chimeric oligonucleotides are notlimited to those with modifications on the sugar, but may also includeoligonucleosides or oligonucleotides with modified backbones, e.g., withregions of phosphorothioate (P═S) and phosphodiester (P═O) backbonelinkages or with regions of MMI and P═S backbone linkages. Otherchimeras include “wingmers,” also known in the art as “hemimers,” thatis, oligonucleotides with two distinct regions. In a preferred exampleof a wingmer, the 5′ portion of the oligonucleotide serves as asubstrate for RNase H and is preferably composed of 2′-deoxynucleotides,whereas the 3′ portion is modified in such a fashion so as to havegreater affinity for the target RNA molecule but is unable to supportnuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted),or vice-versa. In one embodiment, the oligonucleotides of the presentinvention contain a 2′-O-methoxyethyl (2′-O—CH2 CH2 OCH3) modificationon the sugar moiety of at least one nucleotide. This modification hasbeen shown to increase both affinity of the oligonucleotide for itstarget and nuclease resistance of the oligonucleotide. According to theinvention, one, a plurality, or all of the nucleotide subunits of theoligonucleotides may bear a 2′-O-methoxyethyl (—O—CH2 CH2 OCH3)modification. Oligonucleotides comprising a plurality of nucleotidesubunits having a 2′-O-methoxyethyl modification can have such amodification on any of the nucleotide subunits within theoligonucleotide, and may be chimeric oligonucleotides. Aside from or inaddition to 21-O-methoxyethyl modifications, oligonucleotides containingother modifications which enhance antisense efficacy, potency or targetaffinity are also contemplated.

The present invention also provides polynucleotides (for example, DNA,RNA, PNA or the like) that bind to double-stranded or duplex connexinnucleic acids (for example, in a folded region of the connexin RNA or inthe connexin gene), forming a triple helix containing, or “triplex”nucleic acid. Triple helix formation results in inhibition of connexinexpression by, for example, preventing transcription of the connexingene, thus reducing or eliminating connexin activity in a cell. Withoutintending to be bound by any particular mechanism, it is believed thattriple helix pairing compromises the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules to occur.

Triplex oligo- and polynucleotides are constructed using thebase-pairing rules of triple helix formation (see, for example, Cheng etal., J. Biol. Chem. 263: 15110 (1988); Ferrin and Camerini-Otero,Science 354:1494 (1991); Ramdas et al., J. Biol. Chem. 264:17395 (1989);Strobel et al., Science 254:1639 (1991); and Rigas et al., Proc. Natl.Acad. Sci. U.S.A. 83: 9591 (1986)) and the connexin mRNA and/or genesequence. Typically, the triplex-forming oligonucleotides comprise aspecific sequence of from about 10 to about 25 nucleotides or longer“complementary” to a specific sequence in the connexin RNA or gene(i.e., large enough to form a stable triple helix, but small enough,depending on the mode of delivery, to administer in vivo, if desired).In this context, “complementary” means able to form a stable triplehelix. In one embodiment, oligonucleotides are designed to bindspecifically to the regulatory regions of the connexin gene (forexample, the connexin 5′-flanking sequence, promoters, and enhancers) orto the transcription initiation site, (for example, between −10 and +10from the transcription initiation site): For a review of recenttherapeutic advances using triplex DNA, see Gee et al., in Huber andCarr, 1994, Molecular and Immunologic Approaches, Futura Publishing Co,Mt Kisco N.Y. and Rininsland et al., Proc. Natl. Acad. Sci. USA 94:5854(1997).

The present invention also provides ribozymes useful for inhibition ofconnexin activity. The ribozymes bind and specifically cleave andinactivate connexin mRNA. Useful ribozymes can comprise 5′- and3′-terminal sequences complementary to the connexin mRNA and can beengineered by one of skill on the basis of the connexin mRNA sequence.It is contemplated that ribozymes provided herein include those havingcharacteristics of group I intron ribozymes (Cech, Biotechnology 13:323(1995)) and others of hammerhead ribozymes (Edgington, Biotechnology10:256 (1992)).

Ribozymes include those having cleavage sites such as GUA, GUU and GUC.Short RNA oligonucleotides between 15 and 20 ribonucleotides in lengthcorresponding to the region of the target connexin gene containing thecleavage site can be evaluated for secondary structural features thatmay render the oligonucleotide more desirable. The suitability ofcleavage sites may also be evaluated by testing accessibility tohybridization with complementary oligonucleotides using ribonucleaseprotection assays, or by testing for in vitro ribozyme activity inaccordance with standard procedures known in the art.

Further contemplated are antisense compounds in which antisense andribozyme functions can be combined in a single oligonucleotide.Moreover, ribozymes can comprise one or more modified nucleotides ormodified linkages between nucleotides, as described above in conjunctionwith the description of illustrative antisense oligonucleotides providedherein.

The present invention also provides polynucleotides useful forinhibition of connexin activity by methods such as RNA interference(RNAi). This and other techniques of gene suppression are well known inthe art. A review of this technique is found in Science 288:1370-1372(2000). RNAi operates on a post-transcriptional level and is sequencespecific. The process comprises introduction of RNA with partial orfully double-stranded character, or precursors of or able to encode suchRNA into the cell or into the extracellular environment.

As described by Fire et al., U.S. Pat. No. 6,506,559, the RNA maycomprise one or more strands of polymerized ribonucleotide. Thedouble-stranded structure may be formed by a single self-complementaryRNA strand or two complementary RNA strands. The RNA may includemodifications to either the phosphate-sugar backbone or the nucleosides.RNA duplex formation may be initiated either inside or outside the cell.

Studies have demonstrated that one or more ribonucleases specificallybind to and cleave double-stranded RNA into short fragments. Theribonuclease(s) remains associated with these fragments, which in turnspecifically bind to complementary mRNA, i.e., specifically bind to thetranscribed mRNA strand for the connexin gene. The mRNA for the connexingene is also degraded by the ribonuclease(s) into short fragments,thereby obviating translation and expression of the connexin gene, andso inhibiting connexin activity. Additionally, an RNA polymerase may actto facilitate the synthesis of numerous copies of the short fragments,which exponentially increases the efficiency of the system. A uniquefeature of this gene suppression pathway is that silencing is notlimited to the cells where it is initiated. The gene-silencing effectsmay be disseminated to other parts of an organism and even transmittedthrough the germ line to several generations.

In one aspect, the double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing strategy of RNAi involves the use of shortinterfering RNAs (siRNA). The use of the general RNAi approach issubject to certain limitations, including the nonspecific antiviraldefense mechanism in mammalian cells activated in response to long dsRNAmolecules (Gil J, Esteban M, “Induction of apoptosis by thedsRNA-dependent protein kinase (PKR): Mechanisms of action”. Apoptosis2000, 5:107-114). Advances in the field have been made with thedemonstration that synthetic duplexes of 21 nucleotide RNAs couldmediate gene specific RNAi in mammalian cells without invoking genericantiviral defense mechanisms (Elbashir S, et al., “Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammaliancells”. Nature, 411:494-498 (2001); Caplen N. et al., Proc Natl AcadSci, 98:9742-9747 (2001)). Thus, siRNAs are increasingly beingrecognized as powerful tools for gene-specific modulation.

As described herein, RNAi includes to a group of related gene-silencingmechanisms sharing many common biochemical components in which theterminal effector molecule is for example, but not limited to, a small21-23-nucleotide antisense RNA. One mechanism uses a relatively long,dsRNA ‘trigger’; which is processed by the cellular enzyme Dicer intoshort, for example, but not limited to, 21-23-nucleotide dsRNAs,referred to as siRNAs. The strand of the siRNA complementary to thetarget RNA becomes incorporated into a multi-protein complex termed theRNA-induced silencing complex (RISC), where it serves as a guide forendonucleolytic cleavage of the mRNA strand within the target site. Thisleads to degradation of the entire mRNA; the antisense siRNA can then berecycled. In lower organisms, RNA-dependent RNA polymerase also uses theannealed guide siRNA as a primer, generating more dsRNA front thetarget, which serves in turn as a Dicer substrate, generating moresiRNAs and amplifying the siRNA signal. This pathway is commonly used asa viral defense mechanism in plants.

As described herein, the siRNA may consist of two separate, annealedsingle strands of for example, but not limited to, 21-23 nucleotides,where the terminal two 3″-nucleotides are unpaired (3″ overhang).Alternatively, the siRNA may be in the form of a single stem-loop, oftenreferred to as a short hairpin RNA (shRNA). Typically, but not always,the antisense strand of shRNAs is also completely complementary to thesense partner strand of the si/shRNA.

In mammalian cells, long dsRNAs (usually greater than 30 nucleotides inlength) trigger the interferon pathway, activating protein kinase R and2; 5″-oligoadenylate synthetase. Activation of the interferon pathwaycan lead to global downregulation of translation as well as global RNAdegradation. However, shorter siRNAs exogenously introduced intomammalian cells have been reported to bypass the interferon pathway.

The siRNA antisense product can also be derived from endogenousmicroRNAs. In human cells, regardless of the initial form (siRNAs andmicroRNAs) or processing pathway, a final mature for example, but notlimited to, 21-23-nucleotide antisense RNA that is completely homologousto the mRNA will direct mRNA cleavage. In general, the effect ofmismatches between siRNAs and target sites can vary from almost none tocomplete abrogation of activity, for reasons that are only partiallyunderstood; however, in at least one case, partial homology resulted inmRNA translation inhibition. In general, siRNA with target mismatchesdesigned to mimic a prototypical microRNA-target interaction can mediatevarying degrees of translational repression, depending on both thespecific interaction and the number of target sites in the mRNA. RNAican be activated by either exogenous delivery of preformed siRNAs or viapromoter-based expression of siRNAs or shRNAs.

Short interfering RNAs (siRNA) can be chemically synthesized orgenerated by DNA-based vectors systems. In general, this involvestranscription of short hairpin (sh)RNAs that are efficiently processedto form siRNAs within cells (Paddison P, Caudy A, Hannon G: Stablesuppression of gene expression by RNAi in mammalian cells. Proc NatlAcad Sci USA 99:1443-1448 (2002); Paddison P, Caudy A, Bernstein E,Hannon G, Conklin D: Short hairpin RNAs (shRNAs) inducesequence-specific silencing in mammalian cells. Genes & Dev 16:948-958(2002); Sui G, et al., Proc Natl Acad Sci 8:5515-5520 (2002);Brummelkamp T, et al., Science 296:550-553 (2002)). Therefore, in thecontext, siRNAs can be employed as an effective strategy for thetissue-specific targeting and modulation of gene expression.

Oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors known in the art. Any other means for such synthesis may also beemployed; the actual synthesis of the oligonucleotides is wellrecognized in the art. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and 2′-alkoxy or21-alkoxyalkoxy derivatives, including 2′-O-methoxyethyloligonucleotides (Martin, P. Helv. Chim. Acta 78, 486-504 (1995)). It isalso well known to use similar techniques and commercially availablemodified amidites and controlled-pore glass (CPG) products such asbiotin, fluorescein, acridine or psoralen-modified amidites and/or CPG(available from Glen Research, Sterling, Va.) to synthesizefluorescently labeled, biotinylated or other conjugatedoligonucleotides.

Methods

In another aspect, the invention includes methods of treating a subject(e.g. patient), target organ, target tissue, or target cell byadministering compounds provided herein.

Certain embodiments are directed to methods for treating a subject,target organ, target tissue, or target cell with a vascular disordercomprising administering to a subject an antisense compound, mimeticpeptide, or other compound provided herein capable of inhibiting theexpression, formation, or activity of a connexin hemichannel. Vasculardisorders include, for example, any disorder or condition associatedwith arteries, blood vessels, and the vascular and/or cardiovascularsystem, including disorder or conditions in associated organs ortissues. Representative examples include, without limitation,atherosclerosis, microvascular disorders, macrovascular disorders,stroke, cerebrovascular disease (cerebral ischemia), thromboses,vascular injuries resulting from trauma (e.g. subcutaneous wounds, stentinsertion, restenosis, or angioplasty), vascular damage resulting fromelevated levels of glucose (diabetes), diabetic retinopathy, vasculardiseases of the extremities, organ ischemia, and endothelial celldisruption.

Other embodiments are directed to methods of treating an inflammatorydisorder comprising administering to a subject, target organ, targettissue, or target cell an antisense compound, mimetic peptide, or othercompound provided herein capable of inhibiting the expression,formation, or activity of a connexin hemichannel.

Other embodiments are directed to methods for treating a subject, targetorgan, target tissue, or target cell in connection with a transplant orgrafting procedure comprising administration to said patient anantisense compound, mimetic peptide, or other compound provided hereincapable of inhibiting the expression, formation or activity of aconnexin hemichannel and inhibiting or preventing tissue edemaassociated with said transplant or grafting procedure.

Other embodiments are directed to methods of treating myocardialinfarction and its associated heart diseases comprising administering toa subject an antisense compound, mimetic peptide, or other compoundprovided herein capable of inhibiting the expression, formation, oractivity of a connexin hemichannel.

Still other embodiments are directed to methods of treating coronaryartery disease comprising administering to a subject, target organ,target tissue, or target cell an antisense compound, mimetic peptide, orother compound provided herein capable of inhibiting the expression,formation, or activity of a connexin hemichannel.

Other embodiments are directed to methods of treating damage associatedwith ischemia, including initial ischemic damage and a subsequentreperfusion injury.

Other embodiments are directed to methods of treating myocardialinfarction and its associated heart diseases comprising byco-administering to a subject an antisense compound, mimetic peptide, orother compound provided herein capable of inhibiting the expression,formation, or activity of a connexin hemichannel in combination withother known therapeutic approaches or procedures for the treatment ofischemic conditions associated with the heart, including those describedin Baker D. W. et al. (ACC/AHA Guidelines for the Evaluation andManagement of Chronic Heart Failure in the Adult, 2001; American Collegeof Cardiology and the American Heart Association, the contents of whichis hereby incorporated by reference). Such procedures include, forexample, cardiac transplantation, surgical and mechanical approaches,mitral valve repair or replacement for hemodynamic and clinicalimprovements, extra-corporeal devices for circulatory support, leftventricular assist devices, mechanical decompression, leftventriculectomy (Batista procedure), cardiomyoplasty and a variant ofthe aneurysmectomy procedure for the management of patients withischemic cardiomyopathy.

Implants and other surgical or medical devices may be coated with (orotherwise adapted to release) agents of the invention (e.g.anti-connexin compounds and compositions) in a variety of manners,including for example: (a) by directly affixing to the implant or devicean anti-connexin agent or composition (e.g. by either spraying theimplant or device with a polymer/drug film, or by dipping the implant ordevice into a polymer/drug solution, or by other covalent or noncovalentmeans); (b) by coating the implant or device with a substance such as ahydrogel which will in turn absorb the anti-connexin composition (oranti-connexin factor above); (c) by interweaving anti-connexincomposition coated thread (or the polymer itself formed into a thread)into the implant or device; (d) by inserting the implant or device intoa sleeve or mesh which is comprised of or coated with an anti-connexincomposition; (e) constructing the implant or device itself with ananti-connexin agent or composition; or (f) by otherwise adapting theimplant or device to release the anti-connexin agent. Within preferredembodiments of the invention, the composition should firmly adhere tothe implant or device during storage and at the time of insertion. Theanti-connexin agent or composition should also preferably not degradeduring storage, prior to insertion, or when warmed to body temperatureafter insertion inside the body (if this is required). In addition, itshould preferably coat the implant or device smoothly and evenly, with auniform distribution of anti-connexin agent, while not changing thestent contour. Within preferred embodiments of the invention, theanti-connexin agent or composition should provide a uniform,predictable, prolonged release of the anti-connexin factor into thetissue surrounding the implant or device once it has been deployed. Forvascular stents, in addition to the above properties, the compositionshould not render the stent thrombogenic (causing blood clots to form),or en cause significant turbulence in blood flow (more than the stentitself would be expected to cause if it was uncoated).

The anti-connexin compounds, compositions, and methods provided hereincan be used in a variety of procedures that utilize of implants, medicaland surgical devices, and the like. In one aspect, implants, surgicaldevices or stents, are coated with or otherwise constructed to containand/or release any of the anti-connexin agents provided herein.Representative examples include cardiovascular devices (e.g.,implantable venous catheters, venous ports, tunneled venous catheters,chronic infusion lines or ports, including hepatic artery infusioncatheters, pacemaker wires, implantable defibrillators);neurologic/neurosurgical devices (e.g., ventricular peritoneal shunts,ventricular atrial shunts, nerve stimulator devices, dural patches andimplants to prevent epidural fibrosis post-laminectomy, devices forcontinuous subarachnoid infusions); gastrointestinal devices (e.g.,chronic indwelling catheters, feeding tubes, and shunts) opthalmologicimplants (e.g., multino implants and other implants for neovascularglaucoma, drug eluting contact lenses for pterygiums, splints for faileddacrocystalrhinostomy, drug eluting contact lenses for cornealneovascularity, implants for diabetic retinopathy, drug eluting contactlenses for high risk corneal transplants); otolaryngology devices (e.g.,ossicular implants, Eustachian tube splints or stents for glue ear orchronic otitis as an alternative to transtempanic drains); plasticsurgery implants (e.g., prevention of fibrous contracture in response togel- or saline-containing breast implants in the subpectoral orsubglandular approaches or post-mastectomy, or chin implants), andorthopedic implants (e.g., cemented orthopedic prostheses).

An antisense compound, mimetic peptide, or other compound providedherein can be administered at a predetermined time in certainembodiments. In certain embodiments, an antisense connexin hemichannelexpression, formation, or activity is inhibited in endothelial cells. Incertain embodiments, a subject may be treated for a vascular disordercomprising a stroke. In certain embodiments, a subject may be treatedfor a vascular disorder comprising an ischemia. Such an ischemia may be,for example, a tissue ischemia, a myocardial ischemia, or a cerebralischemia. In certain embodiments, a subject treated herein is at risk ofloss of neurological function by ischemia. In other embodiments, asubject may be treated for a vascular disorder comprising treating orameliorating cell death or degeneration in the central or peripheralnervous system that is caused by an ischemia. In certain embodiments, asubject may treated for a vascular disorder where an antisense compound,mimetic peptide, or other compound provided herein is administered inconnection with a vascular or coronary procedure performed on a subject.In other embodiments, an antisense compound, mimetic peptide, or othercompound provided herein is administered during said vascular orcoronary procedure.

Administration of compounds provided herein may be before or subsequentto a selected time point. The “selected time point” may, for example,correspond to the onset of a disorder or condition such as acardiovascular disorder, inflammation, a vascular disorder, or anischemic event, or with performing a medical procedure such as avascular or coronary procedure. Compounds provided herein may (e.g.antisense compounds, mimetic peptide, etc.) for example, may beadministered before, coincident with, or after a selected time point. Incertain embodiments, compounds are administered immediately and up toabout 24 hours subsequent to a selected time point. In otherembodiments, compounds are administered within about 1 hour after aselected time point, within about 2 hours after selected time point,within about 3 hours after a selected time point, within about 4 hoursafter a selected time point, within about 5 hours after a selected timepoint, within about 6 hours after a selected time point, within about 8hours after a selected time point, within about 10 hours after aselected time point, within about 12 hours after a selected time point,within about 14 hours after a selected time point, within about 16 hoursafter a selected time point, within about 20 hours after a selected timepoint, or within about 24 hours after a selected time point. In certainembodiments, a compound provided herein is administered in connectionwith a heart or other surgery performed on a patient. “Selected timepoints,” as referred to herein, include a time of injury, a time ofperforming a procedure, e.g., a heart or vascular procedured, etc. Incertain other embodiments, a compound provided herein is administered inconnection with a medical device for performing a vascular procedure.

In certain embodiments, the vascular disorder treated is selected fromone or more of ischemic stroke, transient ischemic attack, intracerebralhemorage, subarachnoid hemorage, thromboembolic stroke, venousthrombosis, pulmonary embolism, embolic stroke, cerebrovasculardisorder, peripheral occlusive arterial disease, arteriovenousmalformation, and an aneurysm.

In certain embodiments, the vascular disorder treated is associated withone or more of coronary heart disease, coronary vascular disorder,atherosclerotic vascular disease, athersclerotic plaque rupture, and/orthromboembolic, a vascular disorder associated with hypertension,myocardial infarction, angina, ischemic heart disease, aortic disorder,peripheral arterial diseases, fibromuscular dysplasia, moyamo disease,and thromboangiitis.

In certain embodiments, a inflammatory disorder treated is selected fromone or more of arthritis, rheumatoid arthritis (RA), inflammation,destruction or damage of joints, inflammatory disorder, grave's disease,hashimoto's disease, rheumatoid arthritis, systemic lupus erythematosus,sjogrens syndrome, immune thrombocytopenic purpura, multiple sclerosis,myasthenia gravis, scleroderma, psoriasis, inflammatory bowel disease,crohn's disease, ulcerative colitis, sepsis and septic shock, andautoimmune diseases of the digestive system.

In certain other embodiments, a subject, a target organ, a targettissue, or a target cell is treated for a condition that is associatedwith one or more of hemostatis, thrombosis, fibrinolysis, cardiovasculardisease, diabetes mellitus, endocrine disorders affecting the heart,cardiovascular disease associated with pregnancy, rheumatic fever,cardiovascular disorders associated with HIV-infection, hematologicaland oncological disorders associated with heart disease, neurologicaldisorders associated with heart disease, and renal disorders associatedwith heart disease.

In certain other embodiments, a subject, a target organ, a targettissue, or a target cell is treated in association with a transplant orgrafting procedure associated with heart failure, congenital heartdisease, acquired heart disease in children, valvular heart disease,infective endocarditis, cardiomypopathy, tumors of the heart,pericardial heart disease, traumatic heart disease, pulmonary embolism,pulmonary hypertension, cor pulmonale, and athletic heart syndrome,peripheral arterial circulation disorder, vascular disorder affecting anorgan system, vascular disorder affecting the central nervous system,vascular disorder affecting the brain, vascular disorder affecting theretina, vascular disorder affecting the kidney, vascular disorderaffecting and nerves, microvascular disorder, and macrovasculardisorder. The subject can be treated with a transplant or graftingprocedure associated selected from one or more of a heart transplant,kidney transplant, liver transplant, lung transplant, pancreatictransplant, intestinal transplant, or a combined organ transplant. Thesubject can be treated with a transplant or grafting procedure involvesone or more of eye tissue, skin, heart valves, bones, tendons, veins,ligaments, bone marrow transplants, dental or gum tissue, grafting orimplantation associated with cosmetic surgery, grafting or implantationassociated with a hip or joint replacement procedure, and tissuegrafting or implants involving stem cells.

While not intending being bound by or limited to a particular mechanism,in certain embodiments a subject, target organ, target tissue, or targetcell is treated by administration of an anti-connexin compound that iscapable of to binding or modulating a connexon (hemichannel) for thepurpose of achieving a desired effect, including for example one or moreof the following: for the prevention of oedema in the spinal cordfollowing ischaemia or trauma, for the prevention of blood vessel walldegradation in tissues following ischaemia or trauma (e.g. in brain,optic nerve, spinal cord and heart), for the treatment of inflammatoryarthritis and other inflammatory disorders in which oedema andinflammation are symptomatic or in which blood vessel die back occurs asa result of persistent inflammation, for the treatment of sub-acute orchronic wounds to the cornea of the eye in which prevention of bloodvessel die back allows recovery from limbal ischaemia, for the treatmentof sub-acute or chronic wounds to the cornea of the eye as a means totrigger re-epithelialisation, for the treatment of chemical burns in theeye in order to trigger epithelial recovery and to bring about recoveryfrom sub-acute limbal ischaemia, for the treatment of sub-acute orchronic skin wounds or diabetic ulcers in which prevention of continuedblood vessel die back will allow recovery from tissue ischaemia, for thetreatment of chronic skin wounds or diabetic ulcers in which continuedexpression of connexin 43 at the leading edge preventsre-epithelialisation, for the treatment of perinatal ischaemia usingconnexin mimetic peptides delivered directly to ventricles of the brainor via spinal column/cord, for the inhibition or prevention of oedemafollowing perinatal ischaemia using connexin mimetic peptides delivereddirectly to ventricles of the brain or via spinal column/cord, as atreatment for perinatal ischaemia using connexin mimetic peptidesdelivered systemically, as a treatment for stroke or CNS ischaemia usingconnexin mimetic peptides delivered directly to ventricles of the brainor via spinal column/cord, as a treatment for stroke or CNS ischaemiausing connexin mimetic peptides delivered systemically, for theprevention of epileptiform activity in the brain following ischaemia,for the prevention of epileptiform activity in the brain (e.g.epilepsy), for the prevention of tissue oedema using connexon mimeticpeptides or connexin specific antisense polynucleotides, and/or for theprevention of lesion spread, oedema (and rejection) with reperfusionfollowing organ transplantation.

In another aspect, compounds of the invention are administered in anamount desired to modulate the expression, formation or activity of aconnexin, hemichannel, or a gap junction.

While not intending to be bound by or limited to any mechanism andwithout intending any limitation, it is believed that in certainembodiments a target connexin hemichannel is located on a cellcytoplasmic membrane and it may be desirable to effect an inhibition ofundesired transmission of molecules from the cytoplasm of said cell intoan extracellular space. Again, without intending on being limited to anyparticular mechanism, in certain embodiments it may be desirable toadminister a compound provided herein capable of inhibiting orpreventing progressive infarction and reperfusion injury in the heart bymaintaining blood vessel endothelial cell integrity, for promotingreperfusion of blood to damaged tissue in an amount effective to enhancecell survival and/or tissue repair, for maintaining the blood brainbarrier by inhibiting endothelial cell disruption subsequent to a strokeor injury to the central nervous system, for maintenance of vascularintegrity subsequent to tissue damage, or for inhibiting or preventingtissue edema associated with a transplant or grafting procedure.

Pharmaceutical Compositions

In another aspect, the invention includes pharmaceutical compositionscomprising compounds of the invention, including antisense compounds andmimetic peptides.

The antisense compounds provided herein may also include bioequivalentcompounds, including pharmaceutically acceptable salts and prodrugs.This is intended to encompass any pharmaceutically acceptable salts,esters, or salts of such esters, or any other compound which, uponadministration to an animal including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Peptides, for example, can be altered to enhance theirstability in the body. For example synthetic side chains can be addedwhich link to each other resulting in the peptide becoming circular.This circularisation can also be accomplished by linking the N- andC-termini together forming a pro-drug which, after insertion into thebody, is made linear by protease activity. Accordingly, for example, thedisclosure is also drawn to pharmaceutically acceptable salts of thenucleic acids and prodrugs of such nucleic acids. “Pharmaceuticallyacceptable salts” are physiologically and pharmaceutically acceptablesalts of the nucleic acids provided herein: i.e., salts that retain thedesired biological activity of the parent compound and do not impartundesired toxicological effects thereto (see, for example, Berge et al.,J. of Pharma Sci. 66, 1-19 (1977)).

Peptides or variants thereof, can be synthesized in vitro, e.g., by thesolid phase peptide synthetic method or by enzyme catalyzed peptidesynthesis or with the aid of recombinant DNA technology. Solid phasepeptide synthetic method is an established and widely used method, whichis described in references such as the following: Stewart et al., SolidPhase Peptide Synthesis, W.H. Freeman Co., San Francisco (1969);Merrifield, J. Am. Chem. Soc. 85 2149 (1963); Meienhofer in “HormonalProteins and Peptides,” ed.; C. H. Li, Vol. 2 (Academic Press, 1973),pp. 48-267; and Bavaay and Merrifield, “The Peptides,” eds. E. Gross andF. Meienhofer, Vol. 2 (Academic Press, 1980) pp. 3-285. These peptidescan be further purified by fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on an anion-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; ligand affinitychromatography; or crystallization or precipitation from non-polarsolvent or nonpolar/polar solvent mixtures. Purification bycrystallization or precipitation is preferred.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that areavailable in the art. Examples of such substances include normal salinesolutions such as physiologically buffered saline solutions and water.Specific non-limiting examples of the carriers and/or diluents that areuseful in the pharmaceutical formulations of the present inventioninclude water and physiologically acceptable buffered saline solutionssuch as phosphate buffered saline solutions pH 7.0-8.0. Suitablepharmaceutical carriers include, but are not limited to sterile water,salt solutions (such as Ringer's solution), alcohols, polyethyleneglycols, gelatin, carbohydrates such as lactose, amylose or starch,magnesium stearate, talc, silicic acid, viscous paraffin, fatty acidesters, hydroxymethylcellulose, polyvinyl pyrolidone, etc. Thepharmaceutical preparations can be sterilized and desired, mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsiers, salts for influencing osmotic pressure, buffers,coloring, and/or aromatic substances and the like which do notdeleteriously react with the active compounds. They can also be combinedwhere desired with other active substances, e.g., enzyme inhibitors, toreduce metabolic degradation.

Salts of carboxyl groups of a peptide or peptide variant of theinvention may be prepared in the usual manner by contacting the peptidewith one or more equivalents of a desired base such as, for example, ametallic hydroxide base, e.g., sodium hydroxide; a metal carbonate orbicarbonate base such as, for example, sodium carbonate or sodiumbicarbonate; or an amine base such as, for example, triethylamine,triethanolamine, and the like.

N-acyl derivatives of an amino group of the peptide or peptide variantsmay be prepared by utilizing an N-acyl protected amino acid for thefinal condensation, or by acylating a protected or unprotected peptide.O-acyl derivatives may be prepared, for example, by acylation of a freehydroxy peptide or peptide resin. Either acylation may be carried outusing standard acylating reagents such as acyl halides, anhydrides, acylimidazoles, and the like. Both N-acylation and O-acylation may becarried out together, if desired.

Acid addition salts of the peptide or variant peptide, or of aminoresidues of the peptide or variant peptide, may be prepared bycontacting the peptide or amine with one or more equivalents of thedesired inorganic or organic acid, such as, for example, hydrochloricacid. Esters of carboxyl groups of the peptides may also be prepared byany of the usual methods known in the art.

For oligonucleotides, examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

Oligonucleotides provided herein may additionally or alternatively beprepared to be delivered in a “prodrug” form. The term “prodrug”indicates a therapeutic agent that is prepared in an inactive form thatis converted to an active form (i.e., drug) within the body or cellsthereof by the action of endogenous enzymes or other chemicals and/orconditions. In particular, prodrug versions of the oligonucleotides maybe prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivativesaccording to the methods disclosed in WO 93/24510 to Gosselin et al.,published Dec. 9, 1993.

Compounds provided herein may be formulated in a pharmaceuticalcomposition, which may include pharmaceutically acceptable carriers,thickeners, diluents, buffers, preservatives, surface active agents,neutral or cationic lipids, lipid complexes, liposomes, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients and the like in addition to the oligonucleotide.

Pharmaceutical compositions may also include one or more activeingredients such as interferons, antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. Formulations for parenteraladministration may include sterile aqueous solutions which may alsocontain buffers, liposomes, diluents and other suitable additives.Pharmaceutical compositions comprising the oligonucleotides providedherein may include penetration enhancers in order to enhance thealimentary delivery of the oligonucleotides. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e., fattyacids, bile salts, chelating agents, surfactants and non-surfactants(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 8,91-192 (1991); Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems 7, 1-33 (1990)). One or more penetration enhancers from one ormore of these broad categories may be included.

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, recinleate, monoolein (a k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.). Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems page 92 (1991); Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems 7, 1 (1990); E1-Hariri et al., J.Pharm. Pharmacol. 44, 651-654 (1992)).

The physiological roles of bile include the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al McGraw-Hill, New York, N.Y., pages 934-935 (1996)).Various natural bile salts, and their synthetic derivatives, act aspenetration enhancers. Thus, the term “bile salt” includes any of thenaturally occurring components of bile as well as any of their syntheticderivatives.

Complex formulations comprising one or more penetration enhancers may beused. For example, bile salts may be used in combination with fattyacids to make complex formulations. Chelating agents include, but arenot limited to, disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines) [Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems page 92 (1991); Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems 7, 1-33 (1990);Buur et al., J. Control Rel. 14, 43-51 (1990)). Chelating agents havethe added advantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al, Critical Reviews in Therapeutic Drug Carrier Systems page 92(1991)); and perfluorochemical emulsions, such as FC-43 (Takahashi etal, J. Pharm. Pharmacol. 40, 252-257 (1988)). Non-surfactants include,for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al, Critical Reviews inTherapeutic Drug Carrier Systems page 92 (1991)); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 39, 621-626(1987)).

As used herein, “carrier compound” includes a nucleic acid, or analogthereof, which is inert (i.e., does not possess biological activity perse) but is recognized as a nucleic acid by in vivo processes that reducethe bioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. In contrast to a carrier compound, a“pharmaceutically acceptable carrier” (excipient) is a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal. Thepharmaceutically acceptable carrier may be liquid or solid and isselected with the planned manner of administration in mind so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include, butare not limited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodiumstarch glycolate, etc.); or wetting agents (e.g., sodium laurylsulphate, etc.).

The compositions provided herein may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional compatible pharmaceutically-active materials such as,e.g., antipruritics, astringents, local anesthetics or anti-inflammatoryagents, or may contain additional materials useful in physicallyformulating various dosage forms of the composition of presentinvention, such as dyes, flavoring agents, preservatives, antioxidants,opacifiers, thickening agents and stabilizers. However, such materials,when added, should not unduly interfere with the biological activitiesof the components of the compositions provided herein.

Regardless of the method by which compounds (e.g. oligonucleotides,mimetic peptides, etc.) are introduced into a patient, colloidaldispersion systems may be used as delivery vehicles to enhance the invivo stability of the oligonucleotides and/or to target theoligonucleotides to a particular organ, tissue or cell type. Colloidaldispersion systems include, but are not limited to, macromoleculecomplexes, nanocapsules, microspheres, beads and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, liposomesand lipid:oligonucleotide complexes of uncharacterized structure. Apreferred colloidal dispersion system is a plurality of liposomes.Liposomes are microscopic spheres having an aqueous core surrounded byone or more outer layers made up of lipids arranged in a bilayerconfiguration (see, generally, Chonn et al., Current Op. Biotech. 6,698-708 (1995)).

In certain embodiments, antisense compounds and mimetic peptides can beincorporated into or used in conjunction with a biodistributiondirecting moiety, including one or more polymer, to direct thebiodistribution of the antisense compound, mimetic peptide, or othercompound provided herein to the proximity of the a desired target or toallow for continuous release of thereof. Active agents include, forexample, compounds useful for increasing therapeutic efficacy, foroptimizing biodistribution and bioavailability, for reducing tissuedamage, for promoting healing, or for increasing patient comfort;exemplary active agents include vasoactive agents, anesthetics,therapeutic agents for ischemia, growth factors and cytokines.Alternatively, microparticulate or nanoparticulate polymeric bead dosageforms may be used in composition provided herein. Compounds providedherein may be used in combination with an active agent and encapsulatedin a particulate dosage form with a number of ligand or anti-ligandmolecules attached thereto.

In this manner, mimetic peptides, antisense compounds, and othercompounds provided here, alone or in combination with other activeagents, are released at that site over time to provide a sustainedtherapeutic benefit. Sustained release dosage forms are also useful withregard to other active agents useful in the practice of the presentinvention, such as growth factors, cytokines, and the like. Release ofthe active agent from the particulate dosage forms of the presentinvention can occur as a result of both diffusion and particulate matrixerosion. Biodegradation rate directly impacts active agent releasekinetics.

In certain embodiments, controlled release parenteral formulations ofthe mimetic peptide compositions, antisense compounds, and compounds ofthe present invention can be made as implants, oily injections, or asparticulate systems. Particulate systems include microspheres,microparticles, microcapsules, nanocapsules, nanospheres, andnanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres the therapeutic is dispersed throughoutthe particle. Liposomes can be used for controlled release as well asdrug targeting of entrapped drug.

Antisense polynucleotides may be present in a substantially isolatedform. It will be understood that the product may be mixed with carriersor diluents which will not interfere with the intended purpose of theproduct and still be regarded as substantially isolated. A product mayalso be in a substantially purified form, in which case it willgenerally comprise 90%, e.g. at least about 95%, 98% or 99% of thepolynucleotide or dry mass of the preparation.

In certain embodiments, the pharmaceutical composition of the invention,including antisense compounds or mimetic peptides, can be administeredlocally, nasally, orally, gastrointestinally, intrabronchially,intravesically, intravaginally, into the uterus, sub-cutaneously,intramuscularly, periarticularly, intraarticularly, into thecerebrospinal fluid (ICSF), into the brain tissue (e.g. intracranialadministration), into the spinal medulla, into wounds, intraperitoneallyor intrapleurally, or systemically, e.g. intravenously, intraarterially,intraportally or into the organ directly, such as the heart. Anintravenously administered agent becomes bioavailable faster than anagent administered via other routes, therefore generally renderingintravenous administered agents more toxic. Alternatively, intraarterialadministration of the antisense compounds, mimetic peptides, and othercompounds of the invention can be applied to disease targets present inorgans or tissues for which supply arteries are accessible. Applicationsfor intraarterial delivery include, for example, treatment ofliver-related conditions through hepatic artery administration,brain-related conditions through carotid artery administration,lung-related conditions through bronchial artery administration andkidney-related conditions through renal artery administration. Thus, forexample, in certain embodiments the compound is a peptide/polypeptide(e.g. mimetic peptide) for systemic delivery, and in other embodimentsthe compound is a peptide/polypeptide (e.g. mimetic peptide) for directdelivery (e.g. to the ventricles of the brain or into the spinal cord).

For example, U.S. Pat. No. 6,752,987 and US published app. No.20030148968 to Hammond, incorporated by reference herein, which describein vivo delivery for heart disease, which can be accomplished byinjection of the pharmaceutical composition into a blood vessel or otherconduit directly supplying the myocardium or tissue. Preferably, theinjection can be performed by administration into one or both coronaryarteries or other tissue-specific arteries (or by a bolus injection intoperipheral tissue). By way of illustration, for delivery to themyocardium, such injection is preferably achieved by catheter introducedsubstantially (typically at least about 1 cm) within the lumen of one orboth coronary arteries or one or more saphenous veins or internalmammary artery grafts or other conduits delivering blood to themyocardium. Preferably the injection is made in both left and rightcoronary arteries to provide general distribution to all areas of theheart. To further augment the localized delivery of the peptide mimeticsor peptide mimetics in combination with active agent, and to enhancedelivery efficiency, in accordance with the present invention, one caninfuse a vasoactive agent, preferably histamine or a histamine agonistor a vascular endothelial growth factor (VEGF) protein or a nitric oxidedonor (e.g. sodium nitroprusside), into the tissue to be treated, eithercoincidentally with or, preferably, within several minutes before,introduction of the peptide mimetics or peptide mimetics in combinationwith active agents.

In one aspect, methods of spinal administration include but not limitedto, techniques of spinal injections known in the art. General methods ofintra-spinal injection or administration include, for example, epiduralinjections (including caudal block, translumbar, and transforaminalinjections); facet joint injections (including interarticular and nerveblock injections); hardware injections; sacroiliac joint injections, anddifferential lower extremity injections. With most spinal injections, alocal anesthetic (numbing medication) such as, for example, lidocaine(or Xylocalne) or Bupivacaine (Marcaine), is co-injected into a specificarea of the spine.

In certain aspects, the antisense compounds or mimetic peptides can beadministered alone or co-administered in combination with agents usedfor general treatment of stroke and ischemia. These include, forexample, ischema stroke, which is treated by removing obstruction andrestoring blood flow to the brain, and 2) hemorrhagic stroke, whichinvolves the introduction of an obstruction to prevent rupture andbleeding of aneurysms and arteriovenous malformations. In one aspect,treatments for ischemic stroke can include the use of clot-busters, suchas tPA. Generally, tPA is administered within a three-hour window fromthe onset of symptoms. In another aspect, treatment may includepreventative measure with administration ofanticoagulants/antiplatelets. Antiplatelet agents such as aspirin, andanticoagulants such as warfarin interfere with the blood's ability toclot and can be used to prevent stroke onset. In one aspect, treatmentsfor ischemic stroke can include Carotid Endarterectomy, which is aprocedure where a blood vessel blockage is surgically removed from thecarotid artery. In another aspect, treatments for ischemic stroke caninclude Angioplasty/Stents, which involves the use of balloonangioplasty and implantable steel screens called stents to treatcardiovascular disease in which mechanical devices are used to remedyfatty buildup clogging the vessel. For hemorrhagic stroke, surgicaltreatment is often recommended to either place a metal clip at the base,called the neck, of the aneurysm or to remove the abnormal vesselscomprising an Arteriovenous Malformation (AVM). One such example is theEndovascular Procedures, e.g., “coils,” which is less invasive andinvolve the use of a catheter introduced through a major artery in theleg or arm, guided to the aneurysm or AVM where it deposits a mechanicalagent, such as a coil, to prevent rupture.

In certain aspects, the antisense compounds or mimetic peptides can beco-administered as a combined modality for surgical treatment of strokeand ischemia. Surgical interventions for treatment of stroke includes,but is not limited to, conventional surgical modalities for treatment ofstroke, which can be used to prevent stroke, to treat acute stroke, orto repair vascular damage or malformations (for example, AVM) in andaround the brain. These include, for example, carotid endarterectomywhich is a procedure used to remove atherosclerotic plaque from thecarotid artery when this vessel is blocked; andExtracranial/intracranial (EC/IC) bypass; which is a procedure thatrestores blood flow to a blood-deprived area of brain tissue byrerouting a healthy artery in the scalp to the area of brain tissueaffected by a blocked artery.

In other aspects, surgical modalities for stroke or ischemia include,for example, clipping technique, which is useful for treatment of brainaneurysms that cause subarachnoid hemorrhage. Clipping involves clampingoff the aneurysm from the blood vessel, which reduces the chance that itwill burst and bleed. In another aspect, surgical modality can include“detachable coil technique” for the treatment of high-risk intracranialaneurysms. The technique generally involves the insertion of a smallplatinum coil through an artery in the thigh and threaded through thearteries to the site of the aneurysm. The coil is then released into theaneurysm, where it evokes an immune response from the body. The bodyproduces a blood clot inside the aneurysm, strengthening the arterywalls and reducing the risk of rupture. Once the aneurysm is stabilized,a neurosurgeon can clip the aneurysm with less risk of hemorrhage anddeath to the patient. It is also contemplated that the surgicaltreatment of stroke include recently developed techniques such asStereotactic Microsurgery for AVMs and Aneurysms. It employssophisticated computer technology and geometric principles to pinpointthe precise location of the AVM. During the procedure, a custom-fittedframe is attached to the patient's head and three-dimensional referencepoints are established using CT or MRI. This technique allowsneurosurgeons to locate the AVM within one or two millimeters so theycan operate, using microscope-enhanced methods and delicate instruments,without affecting normal brain tissue. Other modalities include, forexample, Stereotactic Radiosurgery for AVMs, which is a minimallyinvasive, relatively low-risk procedure that uses the same basictechniques as stereotactic microsurgery to pinpoint the precise locationof the AVM. Once located, the AVM can be obliterated by focusing a beamof radiation that causes it to clot and then disappear. Due to theprecision of this technique, normal brain tissue usually is notaffected. Other modalities include, for example, Hypothermia, whichutilizes hypothermia (cooling of the body) to prevent stroke duringsurgical treatment of giant and complex aneurysms or difficult AVMs.Dropping the brain temperature gives the surgeon the necessary time tooperate with minimal risk of surgery-induced stroke. Special equipmentknown as a cardiopulmonary bypass machine is sometimes used tocompletely shunt blood flow away from the brain while the body is placedunder deep hypothermia. Other modalities include, for example,revascularization, which is a surgical technique for treating aneurysmsor blocked cerebral arteries. The technique essentially provides a newroute of blood to the brain by grafting another vessel to a cerebralartery or providing a new source of blood flow to the brain.

In certain embodiments, targeted administration can be conducted usingantisense compounds or mimetic peptides alone or in combination withother active agents such as, for example, compounds useful forincreasing efficacy, reducing tissue damage, promoting healing, orincreasing patient comfort. US published patent application 20040259768describes methods and agents for targeted release and the contents ofwhich is hereby incorporated by reference.

A variety of catheters and delivery routes can be used to achieveintracoronary delivery, as is known in the art. For example, a varietyof general-purpose catheters, as well as modified catheters, suitablefor use in the present invention are available from commercial supplierssuch as Advanced Cardiovascular Systems (ACS), Target Therapeutics andCordis. Also, where delivery to the myocardium is achieved by injectiondirectly into a coronary artery (which is presently most preferred), anumber of approaches can be used to introduce a catheter into thecoronary artery, as is known in the art. By way of illustration, acatheter can be conveniently introduced into a femoral artery andthreaded retrograde through the iliac artery and abdominal aorta andinto a coronary artery. Alternatively, a catheter can be firstintroduced into a brachial or carotid artery and threaded retrograde toa coronary artery. Detailed descriptions of these and other techniquescan be found in the art (see, e.g., Topol, E J (ed.), The Textbook ofInterventional Cardiology, 2nd Ed. (W.B. Saunders Co. 1994); Rutherford,R B, Vascular Surgery, 3rd Ed. (W.B. Saunders Co. 1989); Wyngaarden J Bet al. (eds.), The Cecil Textbook of Medicine, 19th Ed. (W.B. Saunders,1992); and Sabiston, D, The Textbook of Surgery, 14th Ed. (W.B. SaundersCo. 1991)).

The compounds provided herein may be administered parentally. It issometimes preferred that certain compounds are combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline. Thecomposition may be formulated for parenteral, intramuscular,intracerebral, intravenous, subcutaneous, or transdermal administration.Uptake of nucleic acids by mammalian cells is enhanced by several knowntransfection techniques, for example, those that use transfectionagents. The formulation which is administered may contain such agents.Example of these agents include cationic agents (for example calciumphosphate and DEAE-dextran) and lipofectants (for example Lipofectam™and Transfectam™).

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated gloves, condoms, and the like may also be useful. Compositionsfor oral administration include powders or granules, suspensions orsolutions in water or non-aqueous media, capsules, sachets or tablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids orbinders may be desirable. Compositions for parenteral administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives. In some cases it may be moreeffective to treat a patient with an oligonucleotide in conjunction withother traditional therapeutic modalities in order to increase theefficacy of a treatment regimen. As used herein, the term “treatmentregimen” is meant to encompass therapeutic, palliative and prophylacticmodalities.

Dosing can be dependent on a number of factors, including severity andresponsiveness of the disease state to be treated, and with the courseof treatment lasting from several days to several months, or until acure is effected or a diminution of the disease state is achieved.Toxicity and therapeutic efficacy of compounds provided herein can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals. For example, for determining The LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. Compounds which exhibit large therapeutic indicesare preferred. While compounds that exhibit toxic side effects may beused, care should be taken to design a delivery system that targets suchcompounds to the site of affected tissues in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography. Dosingschedules can be calculated from measurements of drug accumulation inthe body of the patient. Dosages may vary depending on the relativepotency of individual compounds, including peptide mimetics oroligonucleotides, and can generally be estimated based on EC50 s foundto be effective In vitro and in in vivo animal models.

Suitable dosage amounts may, for example, vary from about 0.1 ug up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides, polypeptides, and compounds provided herein will bespecific to particular cells, conditions, and locations. In general,dosage is from 0.01 mg/kg to 100 mg per kg of body weight, and may begiven once or more daily, weekly, monthly or yearly, or even once every2 to 20 years. In certain embodiments, the dosage may be given fromimmediately post surgery to 24 hours, in another embodiment; the dosageis given from 2 hours and up to 24 hours. Long-acting compositions maybe administered every 3 to 4 days, every week, or biweekly depending onthe half-life and clearance rate of the particular formulation. Personsof ordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the mimetic peptide isadministered in maintenance doses, ranging from 0.01 mg/kg to 100 mg perkg of body weight, once or more daily, to once every 20 years. In thetreatment or prevention of conditions which require connexin modulationan appropriate dosage level will generally be about 0.001 to 100 mg perkg patient body weight per day which can be administered in single ormultiple doses. A suitable dosage level can be about 1 to about 40 mg/kgper day. In certain embodiments, compounds provided herein, includingspecifically antisense compounds or mimetic peptides, are administeredin an amount to achieve in vivo concentrations from about 1 micromolarto about 1 millimolar, from about 10 micromolar to about 500 micromolar,or from about 30 micromolar to about 300 micromolar, and from about 25micromolar to about 300 micromolar final concentration over the damagedsite, and including, about 25 micromolar, or about 160 micromolar, orabout 300 micromolar final concentration over the damaged site, andstill more typically between about 1 micromolar to about 10 micromolar.

In another aspect, peptide inhibitors and mimetic peptides may beadministered to achieve from about 0.1 micrograms per ml to about 1 mgper ml, from about 10 micrograms per ml to about 500 micrograms per ml,or from about 100 micrograms per ml to about 500 micrograms per ml,about 250 micrograms per ml, or about 300 micrograms per ml finalconcentration over the damaged site.

The anti-connexin compound (e.g. peptide mimetic molecules) areintroduced at a number of different concentrations preferably between1×10⁻¹⁰ M to 1×10⁻⁴ M. Once the minimum concentration that canadequately modulate a connexin (including control gene expression isidentified, the optimized dose is translated into a dosage suitable foruse in vivo. Thus, an anti-connexin compound can be administered toachieve at desired concentration in vivo in a particular cell, tissue,or organ of a subject (e.g. a mammal). For example, in certainembodiments a mimetic peptide or other peptide-based anti-connexincompound is administered (e.g. systemically, orally, or parenterally,e.g., IV, etc.) to achieve a final in vivo peptide concentration ofabout 0.1 micromolar 1×10⁻⁷ M), about 1 micromolar (1×10⁻⁶ M), about 2micromolar (2×10⁻⁶ M), about 3 micromolar (3×10⁻⁶ M), about 5 micromolar(5×10⁻⁶ M), about 10 micromolar (1×10⁻⁵ M), about 50 micromolar (5×10⁻⁵M), about 250 micromolar (2.5×10⁻⁴ M, about 500 micromolar (5×10⁻⁴ M),and 1 milimolar (1×10⁻³ M), and 5 milimolar (1×10⁻³ M) or greater. Withsome mimetic peptides an in vivo concentration of between about 1 to 10micromolar (1×10⁻⁶ M to 1×10⁻⁵ M), including about 5 micromolar (5×10⁻⁶M), is desirable. In another aspect, a peptide-based anti-connexincompound is administered directly to a tissue (e.g. ventricles of thebrain of a mammal) in an amount of about 0.1 micromol/kg, 1 micromol/kg,10 micromol/kg, 50 micromol/kg, 250 micromol/kg, 500 micromol/kg, 1000micromol/kg, 5000 micromol/kg. For example, an inhibiting concentrationin culture of 1×10⁻⁷ M translates into a dose of approximately 0.6 mg/kgbodyweight for certain compounds. Levels of an anti-connexin compound(e.g. antisense compound or mimetic peptide molecules) approaching 100mg/kg bodyweight or higher may be possible after testing the toxicity ofthe compound in laboratory animals. It is also contemplated that cellsfrom the vertebrate are removed, treated with the mimetic peptide, andreintroduced into the vertebrate.

Compounds described herein can be used in diagnostics, therapeutics,prophylaxis, and as research reagents and in kits. Since theoligonucleotides of this invention hybridize to nucleic acids encodingconnexin, sandwich, calorimetric and other assays can easily beconstructed to exploit this fact. Provision of means for detectinghybridization of oligonucleotide with the connexin genes or mRNA canroutinely be accomplished. Such provision may include enzymeconjugation, radiolabel ling or any other suitable detection systems.Kits for detecting the presence or absence of connexin may also beprepared.

The compounds of the invention may also be used for research purposes.Thus, the specific hybridization exhibited by the oligonucleotides maybe used for assays, purifications, cellular product preparations and inother methodologies which may be appreciated by persons of ordinaryskill in the art.

Various aspects of the invention will now be described with reference tothe following experimental section which will be understood to beprovided by way of illustration only and not to constitute a limitationon the scope of the invention.

The following Examples are included for illustration and not limitation.

Example 1 Central Nervous System

Damage to the central nervous system can be devastating with enormouslong-term cost to society in patient care. The pathological changes thatoccur in severely injured neuronal tissue share common characteristics.Within 24-48 hours after injury the damage spreads significantlyincreasing the size of the area affected. This spread is propagated bythe gap junction-mediated bystander effect by which gap junctionchannels spread neurotoxins and calcium waves from the damage site tootherwise healthy tissue. Lin, J. H. et al. Nature Neurosci. 1: 431-432(1998). This is, however, also accompanied by inflammatory swellingwhich results in closure of the extracellular space and cell death overthe following 24-48 hours. In fetal brain damage the swelling, forexample, can be tracked as a change in cortical electrical impedancewhich measures cytotoxic edema Reddy, K., et al., Pediatric Research 43:674-682 (1998). This new damage, which is not an immediate result of theinitial insult but subsequent events, occurs between 6 and 48 hoursafter injury, but may be as early as two hours, provides a window ofopportunity for treatment preventing damage spread.

FIG. 1 shows that oedema and swelling occurs even when the cord isexcised from the animal (FIG. 1A). In both cases, the oedema can beblocked using antisense oligodexoynucleotides (ODNs) which preventtranslation of the gap junction protein Connexin 43 (FIG. 1B). In theexcised cord segments, the volume of swelling assessed is significantlydifferent between treated and controls (assessed by measuring area ofswelling viewed from the top −p=0.001).

This swelling observed after CNS damage and blocked using connexinspecific antisense ODNs indicates that gap junction hemichannels arebeing expressed and are opening under pathological conditions leading toa direct pathway between the cell cytoplasm and the extracellular space.Neurons subsequently die. Examination of cells 24 hours after injuryshows vacuolation and membrane inward blebbing (FIG. 2) caused by theuptake of extracellular fluid. As described in the FIG. 3A legends,immunohistochemical labeling of connexin43 using antibodies which bindto the extracellular loop (broad band top left of the connexintopography diagram and labelled Gap7M) and antibodies to the cytoplasmiccarboxyl tail of the protein (broad band at bottom right of connexintopography diagram and labelled GAP1A). The cytoplasmic antibody labelsall connexin43 proteins and is used with a red fluorescently taggedsecondary antibody. The Gap7M antibody can only label exposedextracellular loops of hemichannels and is used with a greenfluorescently tagged secondary antibody. Gap7M is sterically hinderedfrom binding to docked connexons which are forming intact channelsbetween cells and the only connexons which label with both antibodies(and will therefore appear yellow when both the red and green secondaryantibody are colocalised) are existing as hemichannels. FIG. 3B: Asdescribed in the Figure legends, this image shows dual labeling with thetwo antibodies described in FIG. 3A applied to spinal cord sections 24hours after a crush wound. The image has small bright spots which arelabelled connexins and large, lighter shaded spots marking cell nucleilabelled with DAPI. A significant portion of the connexin labelling incombined images indicates that the two antibodies (Gap7M and GAP1A) arecolocalised. This means that the connexin extracellular loops areexposed and most of the connexons present have not docked with theneighboring cell's connexons and remains as hemichannels. FIG. 3C: Asdescribed in the Figure legends, this image shows dual labeling with thetwo connexin antibodies described in FIG. 3A applied to spinal cord 24hours after a crush wound. In this case the application of Connexin43specific antisense ODNs has been applied to prevent protein translation.Hemichannels will appear as bright spots (due to colocalisation of greenfluorescently tagged Gap7M and red fluoresecently tagged GAP1A). In theFigure, the larger, lighter spots are cell nuclei labelled with DAPI.Little gap junction protein is labelled, and few hemichannels are seenin these treated cords.

Furthermore, gap junction antibodies which bind and label theextracellular loops of the connexin protein (Gap7M antibodies —FIG. 3A)were shown to label extensive protein levels 24 hours following ratspinal cord injury (FIG. 3B). These antibodies only bind to the portionsof hemichannels that interact to form a multimer upon docking in themembrane. These data indicates that much of the connexin 43 upregulationseen in early stages after CNS damage remains in hemichannel form.

The expression of the connexin protein and hemichannel formation isblocked using connexin 43 specific antisense ODNs applied in a PluronicF-127 (Poloxamer) gel at the time of wounding (FIG. 3C).

This treatment indicates that ODN treatment in an explant model hasmaximum effect on protein levels (maximum knockdown) at 6-8 hours afterapplication with knockdown apparent within 2 hours and protein levelsrecovering after about 24 hours (see Qiu et al, 2003; Becker et al,1999). In the brain similarly, slices of tissue placed into cultureswell, but the swelling can be blocked using the connexin 43 specificantisense ODNs preventing hemichannel formation (C Green—data notshown).

Following injury, upregulation of connexin levels leads to hemichannelformation causing cellular oedema and death. This is not, however,restricted to the neural population. In the excised spinal cordsegments, Isolectin B4 label (which binds to carbohydrates on thesurface of microglial cells and endothelial cells of blood vessels)outlines blood capillaries even after 5 days in culture in the antisensetreated tissue (FIG. 5). In control segments few capillaries remainafter two days (and after 5 days the predominant Isolectin B4 labelingis of activated macrophage phenotype (foam) glial cells). In connexin 43antisense treated brain slices, capillaries remain intact even after twoweeks in culture; while none remain in control slices (data not shown).

Following injury to rat spinal cord in vivo, rats were treated withconnexin specific antisense ODNs and 24 hours later injectedfluoresceinated—Bovine Serum Albumin (FITC tagged BSA) into the rat-tailvein. Control animals show extensive leakage of the dye from thevascular system into surrounding tissues even 5 mm rostral to the woundsite (FIG. 5A). In sharp contract, antisense treated animals show littlesign of leakage with the dye restricted to the capillary bed (FIG. 5B).The capillary endothelial cells, which express connexin 43, are alsoforming hemichannels and becoming disrupted. Importantly, the connexin43 specific antisense ODN treatment provided herein prevents thebreakdown of the blood-brain barrier, breakdown of the vascular system(necessary for reperfusion and recovery), and the spread of damage. Theresults show that this breakdown in the capillary/blood vessel system isnot restricted to the central nervous system, and that a broader rangeof applications, such as for treatment vascular conditions, wouldbenefit from modulation of connexins. As described in the FIG. 5Alegends, this image shows triple labeling of sheep heart ventricle wall24 hours after an ischemic infarct. The four panels comprising thisimage are from tissue distant to the infarcted region. The tissue islabeled with Isolection B4 (top left panel) which is binding to bloodvessel endothelial cells, and with antibodies to Gap7M (top right panel)and antibodies to connexin43 (bottom left panel). The Gap7M antibodiesrecognize conserved extracellular loop regions of the connexin proteinsso are not connexin isoform specific, but do mark hemichannels (they aresterically hindered from accessing their epitope in intact channels).The top left image shows normal capillary structure is present in thisregion. No hemichannels are present (top right) but connexin43 ispresent in intercalated disks of the working myocardium (bottom left).The bottom right panel shows an overlay of the other three images.Little connexin43 label overlies the capillary vessel walls as it ispredominantly associated with the muscle cells. As described in the FIG.5B legends, this image shows triple labeling of sheep heart ventriclewall 24 hours after an ischemic infarct. The region seen is away fromthe infarct but closer to it than is shown in FIG. 5B shows the tissueis labeled with Isolection B4 (top left panel) which is binding to bloodvessel endothelial cells. Most of the vessels are still intact but thevessel walls are disrupted in areas (areas of broader dispersedlabelling). Antibodies to Gap7M (top right panel) label hemichannels.Elongated patches of dense hemichannel label are evident. The bottomleft panel shows antibody labelling of connexin 43. Careful comparisonbetween these first three panels, or analysis of the patterns present onthe bottom right panel which shows the other three merged, shows thatthe connexin43 is uniquely associated with muscle cells. However, thehemichannel antibody has colabelled regions of the blood vessel wallthat appear disrupted, indicating the presence of connexon hemichannelsin those areas. As described in the FIG. 5C legends, this image showstriple labeling of sheep heart ventricle wall 24 hours after an ischemicinfarct. The region shown is within the infarcted area itself. Thetissue is labeled with Isolection B4 (top left panel) which is bindingto blood vessel endothelial cells. Most of the vessels appear disrupted(areas of broader dispersed labelling in discontinuous lines).Antibodies to Gap7M (top right panel) label hemichannels. Multiplepatches of dense hemichannel label are evident across the whole panel.The bottom left panel shows antibody labelling of connexin43. Carefulcomparison between these first three panels, or analysis of the patternspresent on the bottom right panel which shows the other three panelsmerged, shows that the connexin43 is associated with muscle cells, butthe label is in short patches, quite unlike the usual labelling ofconnexin43 in the intercalated disks of cardiac muscle. This indicatesthat the myocytes have also become severely damaged in this centralinfarct region. The hemichannel antibody has colabelled extensiveregions of the blood vessel wall that appear disrupted, indicating thepresence of connexon hemichannels in the blood vessel wall. Fewcapillaries remain intact apparently following this hemichannelexpression. Here, as in FIG. 6B, the Gap7M antibody label does notcolocalise with the connexin43 label (as it does in the spinal cord—FIG.2B), indicating they must be of a different gap junction proteinisoform, most likely connexin45 (See Camelliti, P., et aI., Cardiovasc.Res. 62: 414-425 (2004).

Example 2 Cardiovascular System

This Example examines the regulation and role of gap junctionhemichannels to maintain vascular integrity in the capillary bedadjacent to ischemic tissue damage. A sterile gel foam was deliveredthrough the left anterior descending or circumflex artery to inducetransmural myocardial infarcts. The gel was delivered essentiallyaccording to the methods of Devlin, G., et al., J. An ovine model ofchronic stable heart failure. J. Card Fail. 6: 140-143 (2000).Isolection-B4 labeling of capillary endothelial cells shows that thecapillary bed adjacent to ischemic tissue is breaking down. We haverecently analyzed progressive infarction in the sheep infarct model(Camelliti et al., Spatially and temporally distinct expression offibroblast connexins after sheep infarction, Cardiovascular Research,62:415-425 (2004), and we proposed that this is caused by a gap junctionmediated bystander effect. The data presented herein indicate a key roleof gap junction mediated bystander effect associated with endothelialcell disruption and following expression of gap junction hemichannels.In triple labeling of24-hour ischemic sheep heart using Isolectin-B4,connexin 43 and hemichannel antibodies the data show that thehemichannels are not connexin 43 in this case, but instead appear to beconnexin 45 (FIGS. 6A, 6B, 6C). Note that Gap7M antibody recognizesconserved regions of the first extracellular loop of the connexinprotein and is not connexin specific; it cross reacts with a number ofthe connexin family members. Connexin 45 is the first connexin to beupregulated following ischemic heart injury (Camelliti et al., 2004).This series of panels (FIGS. 6A, 6B, 6C) show that while damage to thevessel walls is not apparent distant from the infarcted region (FIG. 6A)it becomes progressively worse closer to the infarct region (FIG. 6B)and in conjunction with hemichannel expression. Within the infarctregion itself (FIG. 6C) hemichannel protein expression is high, thecapillary walls are extensively disrupted, and myocyte intercalateddiscs (where the connexin 43 gap junctions are located) are becomingdispersed. As described in the FIG. 6 legends, this image shows showsIsolectin B4 (top panel) marking capillary endothelial cells andmyomesin antibody labeling (middle panel) marking M lines in thesacromeres of myocytes in a sheep heart ventricular infarct, 24 hoursafter ischemia. This region is the same as that shown in FIG. 6C. Theblood capillaries are completely disrupted and normal myocyte sarcomericbanding pattern has been destroyed indicating muscle cell death isoccurring in parallel with vessel wall disintegration. The lower imageis a merger of the top two showing the relationship between thedisrupted capillaries and abnormal muscle band labelling.

FIG. 7 shows Isolectin B4 label of disrupted blood vessels within theinfarct zone correlating with sarcomere disruption illustrated usingantibodies for myomesin which label the M-bands of the sarcomeres. As inneural tissues, subsequent damage to the blood vessel walls appears tofollow hemichannel expression, and cell death in general becomessignificant and as a result of hemichannel opening.

It has been reported that in hearts made hypoxic for 30 minutes andreperfused with heptanol (a non-specific gap junction channel blocker)in the medium prevented the oxygen paradox leading to hypercontractionand myocyte death. Garcia-Dorada et al., Circulation 96:3579-3586(1997). These authors reported that hypercontracture may be transmittedto adjacent myocytes through gap junctions. Our data is consistent withthe idea that within 30 minutes hemichannel expression may be playing asignificant role in hypercontracture.

Increased gap junction protein expression and hemichannel opening underpathological conditions is leading to endothelial cell disruption andbreakdown of the cardiac vascular system in the regions surroundingischemia damaged tissue. This finding, first described herein, it isbelieved to have enormous significance for the treatment of reperfusioninjury and is a probable mechanism for progressive infarction. Robbins,S. and Cotran, R. 1979. Pathologic basis of disease. 2^(nd) Edition, WBSaunders Company, Philadelphia.

Example 3 Mimetic Peptide Design

In this example, nine overlapping peptidomimetics were designed to havethe same amino acid sequence as connexin 43 extracellular loop regionsbelieved to be involved in the connexon docking process (Foote et al., JCell Biol 140(5):1187-97, (1998)). These particular peptides were alldesigned to be 11-13 residues long. Some peptides included amino acidsmatching the outer portions of the alpha helical transmembrane subunits,which may show enhanced functional inhibition. Not all of these peptidesare necessarily connexin 43 specific due to the conservation of connexinsequences in the extracellular loop regions.

Peptides targeted to connexon 43 (hemichannel) are shown below. M1, 2, 3and 4 refer to the 1^(st) to 4^(th) transmembrane regions of theconnexon 43 protein respectively. E1 and E2 refer to the first andsecond extracellular loops respectively:

FEVAFLLIQWI (SEQ ID NO:32) M3 & E2 LLIQWYIGFSL (SEQ ID NO:33) E2SLSAVYTCKRDPCPHQ (SEQ ID NO:34) E2 VDCFLSRPTEKT (SEQ ID NO:35) E2SRPTEKTIFII (SEQ ID NO:36) E2 & M4 LGTAVESAWGDEQ (SEQ ID NO:37) M1 & E1QSAFRCNTQQPG (SEQ ID NO:38) E1 QQPGCENVCYDK (SEQ ID NO:39) E1VCYDKSFPISHVR (SEQ ID NO:40) E1

Example 5 Functional Testing of Mimetic Peptides

Two functional tests were carried out using peptides. These functionaltest were (i) blockage of dye (Lucifer Yellow) uptake by cells in spinalcord slices, and (ii) prevention of oedema in spinal cord segments(using connexin 43 specific antisense as a positive control). Allpeptides used were synthesised by Sigma-Genosys (Australia).

Blockage of Dye (Lucifer Yellow) Uptake by Cells in Spinal Cord Slices.

Lucifer Yellow is a small water soluble, fixable, dye able to pass fromcell to cell via gap junction channels, but not across the cellmembrane. The addition of Lucifer Yellow to the extracellular mediummakes it is possible to check for the presence of open gap junctionhemichannels. The dye will appear in the cytoplasm of cells which areexpressing open channels.

Wistar p7 rats were anesthetized with carbon dioxide and immediatelydecapitated. The spinal cord was excised and transferred to cold Hank'sbalanced salt solution (HBSS) at pH 7.4. Excess branch nerves andligaments were removed and the cord transferred to a manual tissuechopper and a series of 500 micron thick slices cut. The damage causedby the slicing induces connexin 43 upregulation through the entire slice(exacerbated by the gap junction mediated bystander effect), and leadsto the expression of connexin hemichannels. Slices were placed onto 3 cmdiameter Millipore inserts in 24 well plates and cultured in thepresence of mimetic peptides in the media. The final concentration forall 9 peptides tested was 500 micromolar. Controls were; no peptideadded, or with 1% ethanol or 1% DMSO added as some peptides werere-dissolved in these compounds (peptides were received lyophilised).Some slices were also treated at this time with connexin 43 specificantisense oligodeoxynucleotides in 30% Pluronic F-127 gel or with gelonly, as control experiments. The slices treated with antisenseoligodeoxynucleotides indicated that the antisense prevented connexinexpression and subsequently dye uptake, and thus these acted as apositive control.

Slices were incubated for four hours at 37 degrees C., and then 2.5 mgper ml Lucifer Yellow was added to each well for 30 minutes (in thedark). The tissues were then rinsed twice in PBS, with three further 10minute washes, and the slices fixed with 4% paraformaldehyde. They werethen viewed using a Leica TCS4D laser scanning confocal microscope toassess dye uptake into cells, or not.

Results showed that media alone cultured slices, and DMSO, ethanol andgel only treated slices had significant dye uptake. Connexin 43 treatedslices had no dye uptake. The peptide treated slices showed considerabledye uptake, with the exception of those treated with the followingpeptides (which have overlapping sequences):

VDCFLSRPTEKT, (SEQ ID NO:35) and SRPTEKTIFII (SEQ ID NO:36)

The level of dye uptake for slices treated with the peptides having SEQID NOS:32-34 ((FEVAFLLIQWI (SEQ ID NO:32), LLIQWYIGFSL (SEQ ID NO:33),SLSAVYTCKRDPCPHQ (SEQ ID NO:34)) and SEQ ID NOS:37-40 (LGTAVESAWGDEQ(SEQ ID NO:37), QSAFRCNTQQPG (SEQ ID NO:38), QQPGCENVCYDK (SEQ IDNO:39), and VCYDKSFPISHVR (SEQ ID NO:40)) was comparable with controlslices.

In summary, these data show that the spinal cord slices expresshemichannels that are open within 4 hours of injury. Importantly, thedata also show that peptides corresponding to SEQ ID NO: 35 and 36 arecapable of preventing and/or blocking and/or closing the opening of thehemichannels and preventing swelling.

Prevention of Oedema in Spinal Cord

The system described in Example 1 was used to examine the effects of thepeptides VDCFLSRPTEKT (SEQ ID NO:35) and SRPTEKTIFII (SEQ ID NO:36) oncultured spinal cord segments to test their ability to block swelling.

The peptide QQPGCENVCYDK (SEQ ID NO:39) was used a negative controlbecause it allowed dye uptake in the slice cultures described above, andwas thus believed to not be able to block oedema in the segments. DMSOwas again used as an additional control.

5 mm long spinal cord segments were placed in separate wells of a 24well plate in HBSS. The segments were held to the bottom of the wellusing a small drop of Superglue. The HBSS was removed and 500 micromolarpeptide (final concentration) added to media (no peptide for media aloneor DMSO controls). The plates were incubated for 24 hours, the mediaremoved and the tissue fixed with Bouin's fixative for 24 hours.Analysis involved photographing cord segments from above, with Image Jused to calculate the total area of the cord segment compared with thearea of swelling at the cut ends of the segments. Swelling (oedema) wascalculated as (cultured area—original area divided by original area) togive % swelling. Single factor Analysis of Variance was used todetermine statistical significance, with cut-off level for significanceat p=0.05.

Results were that DMSO treated cord segments swelled the most (33%) withall control cord segments swelling 21-23%. Segments treated with thepeptide QQPGCENVCYDK (SEQ ID NO:39) also showed 23% swelling butpeptides VDCFLSRPTEKT (SEQ ID NO:35) and SRPTEKTIFII (SEQ ID NO:36)showed a reduced 15 and 17% swelling respectively (FIG. 8). Thedifference between the peptides VDCFLSRPTEKT (SEQ ID NO:35) andSRPTEKTIFII (SEQ ID NO:36) treated cord segments and controls wassignificant (p=0.43). Subsequent histological examination of the tissuesrevealed that the peptide SRPTEKTIFII (SEQ ID NO:36) treated segmentsretained better morphology and so dose response experiments were thencarried out with this peptide.

Determining the most effective concentration of the peptide SRPTEKTIFII(SEQ ID NO:36) for blocking of oedema in spinal cord segments wascarried out using the same protocol. In this case a dose response wasdetermined with final concentration of peptides used at 5, 10, 50 250and 500 micromolar. Results are shown in FIG. 8. Interestingly thelowest concentration of the peptide (5 micromolar) gave the best result(least oedema) when compared to media alone (p=0.001). The middle range50 micromolar was somewhat less effective in repeat experiments.

Immunohistochemical analysis showed reduced astrocytosis (GFAPexpression) in the peptide SRPTEKTIFII (SEQ ID NO:36) treated segmentsafter 24 hours in culture. Again 5 micromolar was most effective atpreventing the inflammatory response although difference between theconcentrations used were less marked than in the oedema experiments. Alltreatments showed significantly reduced glial fibrillary acidic protein(GFAP) expression (area of label per area of section analysed usingImage J) (Table 7).

Our experiments have indicated that a dose of 5 μmol/kg brain weight forthe perinatal sheep experiment (average 25 g at this fetal age) of theselected peptidomimetic (SEQ ID NO:36) given in 1 ml of artificial CSFi.c.v. (vs. vehicle of CSF alone) over one hour, followed by a further 1ml per day perfused into the brain for 72 hours had a significanteffect. Dosages of about 5 μmol/kg brain, 50 μmol/kg and 250 μmol/kgbrain weight are also possible. For intravenous (systemic delivery) theeffect of log-order increases in plasma concentrations can be used todetermine an appropriate dose, starting with loading doses to achieve0.5 μmol/L (mean fetal blood volume is approximately 350 ml in ourperinetal sheep at this age), then 5, 10, 50, 250, 500 and 5000 μM.

ImageJ is Java script open source, public domain image analysis softwareoriginally developed by the NIH (and called NIH Image).

TABLE 7 Treatment Area of GFAP label (square units) Control 2450  5micromolar peptide 5 300  50 micromolar peptide 5 950 250 micromolarpeptide 5 1000 500 micromolar peptide 5 750

Table 7: Areas of GFAP label in images taken in spinal cord 24 hoursafter slicing. Control cords have high levels of GFAP indicating aninflammatory response and greater bystander effect than the treatedsegments. The lower peptide concentration is the most effective atlimiting astrocytosis.

Activated microglial cell counts revealed no differences at 24 hours asexpected. This secondary inflammatory process (differentiation andproliferation from resting microglial cells to macrophage phenotype)usually takes three-seven days.

Example 6 In Vivo Application of Connexin Specific Mimetic Peptides toBlock Ischaemia and Epileptiform Brain Activity in a Perinatal SheepModel

Brain damage resulting from cerebral ischaemia remains a significantproblem at all stages of life. In the term newborn, moderate to severedamage at birth occurs in 2 to 3 per 1000 live births. One of the moststriking features is that the injury spreads over time from the mostseverely damaged areas outwards, into previously undamaged regions.Immediately after cerebral ischaemia there is transient recovery ofbrain metabolism that lasts for some hours. After this, however, thereis a progressive mitochondrial failure, coupled with secondary cellswelling, reaching a maximum 36 to 48 h after initial injury.

Active coupling of gap junctions, between glia and neurons, mediates abystander effect in which cell death signals are transferred from dyingcells to less severely injured or healthy cells. Earlier studies showedthat in vivo topical application of gap junction protein connexin 43specific antisense oligodeoxynucleotides can restrict the spread ofinjury and secondary inflammation following trauma. See WO2000/44409 toBecker, D. and Green. C., entitled “Formulations Comprising AntisenseMucleotides to Connexins.”

The peptide SRPTEKTIFII (SEQ ID NO:36) was used in an in vivo model ofsheep perinatal ischaemia. The data indicate that connexin specificmimetic peptides provide a treatment with potential to significantlyreduce secondary damage in the ischaemic perinatal brain or following astroke. Preliminary analysis showed that 24 h after cerebral ischaemiain the near-term fetal sheep there is increased expression of gapjunction hemi-channels (i.e. uncoupled connexons) (FIG. 9).

A Romney-Suffolk cross fetal sheep was instrumented between 117 to 124days of gestation (0.85 term) under general anaesthesia as describedelsewhere in detail (Gerrits et al, Pediatr Res 57(3):342-6, (2005);Guan et al., Neuroscience 95(3):831-839, (1999); Guan et al., J CerebBlood Flow Metab 21(5):493-502, (2001); Gunn et al., J Clin Invest99(2):248-256, (1997), Gunn et al., Pediatrics 102(5):1098-1106, (1998),Gunn et al., Pediatr Res 46(3):274-280, (1999); Roelfsema et al., JCereb Blood Flow Metab 24(8):877-886, (2004)). Instrumentation includedbrachial artery and vein catheters, EKG electrodes, an inflatableoccluder around a fetal carotid artery (Gunn et al., 1997; Roelfsema etal., 2004), parietal EEG electrodes 5 and 15 mm anterior, and 10 mmlateral to bregma, a pair of electrodes placed lateral to these tomeasure cortical impedance (a measure of cytotoxic oedema (Gunn et al.,1997) and a 17-mm-long left i.c.v. cannula 4 mm anterior and 6 mmlateral to bregma. The instrumentation was exteriorised to the maternalflank, uterine and abdominal walls closed, and fetal vascular cathetersheparinised (20 IU/ml). The maternal wound was infiltrated with a longacting local anaesthetic bupivacaine (100 mg/20 ml).

After 5 days recovery, fetal cerebral hypoperfusion was induced by a 30minute period of bilateral carotid artery occlusion (Gunn et al., 1997;Roelfsema et al., 2004; Tan et al., Ann Neurol 32(5):677-682, (1992);Tan et al., Pediatr Res 39(5):791-797, (1996)). Anintracerebroventricular infusion of connexin mimetic peptide 5 wasstarted 90 minutes after the ischaemia and continued for 72 hours. Adose of 5 μmol/kg brain weight (average 25 g at this fetal age) of thepeptidomimetic 5 was given in 1 ml of artificial CSF i.c.v. (vs. vehicleof CSF alone) over one hour, followed by a further 1 ml per day perfusedinto the brain for 72 hours. The experiment was ended by a maternalintravenous overdose of sodium pentobarbital (30 ml, 300 mg/ml). Thefetal brain was removed for histology and immunohistochemical analysis.

The results (FIG. 10) show that early infusion of the peptide attenuatessecondary, delayed seizure activity and cytotoxic oedema.

In summary, these data demonstrate that a peptidomimetic protein thattargets the extracellular domain of connexin 43 hemichannels cansuppress secondary oedema and inflammation following brain ischaemia. Ina near-term fetal sheep, we found that cerebral ischaemia was associatedwith a dramatic induction of connexin 43 and of hemichannels within 24 hafter ischaemia, while an i.c.v infusion of the peptidomimetic proteinto the term fetus from 90 min after reperfusion showed significantattenuation of secondary seizures and cytotoxic oedema.

Example 7 Treatment of a Human Patient with Connexin 43 SpecificAntisense In a Sub-Acute Wound—Prevention of Blood Vessel Die BackAllows Recovery from Limbal Ischaemia

In this study, a patient presented with a sub-acute non-healing wound(chemical burn) to the eye. The eye remained inflamed and limbalischaemia was still present after 8 days (indicating poor limbalvascularisation). The limbus contains the stem cells necessary forepithelial recovery of the cornea. Following treatment with connexin 43specific antisense the limbal ischaemia had gone within 20 hours andre-epithelialisation had commenced. The conclusion is that continuinginflammation leads to a persistent die back of the blood vesselsexacerbating the injury through limbal ischaemia. Treatment of the eyewith the connexin 43 antisense reduced the inflammatory response andtriggered epithelial recovery (Qiu et al., Curr Biol 13:1697-1703,(2003)). Note however that this was a sub-acute wound implying treatmentfor chronic wounds is possible—such wounds in humans retain highconnexin 43 levels at the epithelial leading edge (Brandner et al., JInvest Dermatol 122:1310-1320, (2004)). In addition, the treatmentallowed blood vessel recovery. We propose that the mechanism involved isprevention of further hemichannel expression in the vessel wall,allowing vessel regrowth.

Patient: Patient was a 25 year old male. He first presented withalkaline burns to left eye following a building site accident with ahigh pressure concrete hose (concrete/alkali in the eye, coupled withdelay getting to first treatment). The damaged eye had no remainingepithelium covering the front of the eye (including the entire cornea).

Initial Treatment: Patient was put onto 10% ascorbate drops, 10% citratedrops, 1% prednisone acetate (steroid) drops, 1% cyclopentalate andchloramphenicol, plus oral vitamin C and deoxycycline. The prednisonewas delivered hourly for first five days, after which the dose wasreduced to four times per day.

On day four an amniotic membrane was stitched over the cornea.

Connexin 43 Antisense Treatment (DAY ZERO): At day eight post-injury thepatient still had high degree of inflammation, limbal ischaemia, and nosign of epithelial recovery. Ethical permissions were obtained based onlack of any viable treatment alternatives that could save the patient'seye. The other eye has signs of keratoconus and was thus not suitablefor limbal transplant at a later date. The injured eye would either havebeen surgically removed or allowed to become a “conjunctive eye”(wherein the conjunctiva, in the form of a white sheath, grows over theeye to render the patient blind).

Connexin 43 antisense in 30% F-127 Pluronic gel was injected with acatheter needle under the amniotic membrane in two places either side ofthe cornea Approximately 100 microliters of two micromolar anti-connexin43 was injected and gently spread around the cornea using a cotton wandover the amniotic membrane. The gel was injected cold and setimmediately to a soft jelly-like substance.

The patient was removed from all other treatments for eight hours toavoid any potential adverse effects on the treatment. The patient wasthen placed back on steroid drops (three times per day), cyclopentalate(once per day), and ascorbate, citrate, chloramphenicol drops (fourtimes each per day).

Connexin 43 Antisense Treatment (DAY ONE): Within 20 hours followingconnexin 43 antisense treatment the eye had become substantially quieter(reduced inflammation) and the epithelium was growing back in threeplaces. The limbus was well vascularized with good blood flow, and nosign of limbal ischaemia, i.e., there was full blood flow back to thelimbus within 20 hours post-treatment.

Connexin 43 Antisense (DAY THREE): Within 72 hours after connexin 43antisense treatment the patient had continued to improve. The eye wasquiet, the limbal blood supply was excellent, and the epithelium wasgrowing back around 360 degrees. On one side there appeared to be asmall area of lamellapodial crawling, but on the remainder of thecircumference of the cornea there was nice even inward growth.

Connexin 43 Antisense Treatment (DAY SIX): Within six days followingtreatment (14 days post-injury) the epithelium was fully recovered(completely grown over) although it appeared slightly granular in placesand perhaps patchy or thin in places (assessed looking through theamniotic membrane). The limbal region remained well vascularized withfull blood flow.

Forty days after treatment the patient had excellent recovery for achemical burn, showing 6/48 vision unaided and 6/15 pinhole. Two thirdsof the epithelium was absolutely healthy, one third at periphery showingsome conjunctival growth but not covering the pupil and notvascularised. Very good limbal vascularisation.

Optic Nerve Neuropathy

Ischaemic optic neuropathy (ION), also known as stroke of the opticnerve, is a collection of diseases that affects the blood supply to theoptic nerve. ION can be categorised based on the locality or aetiology.Anterior ION (AAOIN) referss to diseases affecting nerve segments priorto lamina cribrosa while the opposite is true for Posterior ION (PION)(Buono et al., Survey of Opthalmology 50:15-26, (2005); Collignon etal., Opthalmology 111:1663-1672, (2004)). PION is less commonlyobserved, and is believed to be caused by infarction of the intraorbitalportion of the optic nerve, most likely due to giant cell arteritis(GCA) or as a secondary complication of surgical procedures (Buono andForoozan, 2005; Ho et al., Journal of Neurosurgical Anesthesiology17:38-44, (2005)). ION can be also be divided into arteritic (ArteriticION) and non-arteritic (NAION) based on aetiology. Arteritic ION isalways caused by GCA and usually results in thrombotic occlusion of theposterior ciliary artery, which can lead to concomitant obstruction ofother arteries in the optic nerve (Galasso et al., Seminars inOpthalmology 19:75-77, (2004)). NAION is the most common form ofnon-glaucomic optic neuropathy with an annual incidence of 2.3/10,000(Collignon-Robe et al., Opthalmology 111:1663-1672, (2004)). GCA is achronic vasculitis of large and medium vessels in the braincharacterised by an increased inflammatory giant cell count (Buono etal., Survey of Opthalmology 50:15-26, (2005); Khosla et al., Journal ofPostgraduate Medicine 50:219-221, (2004); Penn et al., AutoimmunityReviews 2:199-203, (2003)). Visual loss is not common but does occur asa secondary complication owing to occlusion of the anterior vesselssupplying the optic nerve, and is often irreversible (Khosla et al.,2004). The incidence of GCA in Western countries range from 1˜30/10,000with much higher prevalence in the population over 50 years of age (Pennand Dasgupta, 2003).

The typical outcome of ION is the degeneration of axon tracts,accompanied by deterioration or even loss of vision (Buono et al., 2005;Khosla et al., 2004; Penn and Dasgupta, 2003).

Conventional treatments of ION include administration of corticosteroidsand antiplatelet agents (Arnold et al., Seminars in Opthalmology 17:39-46, (2002)). but patients treated with these drugs have notdemonstrated significant improvement from the disease.

In the optic nerve both astrocytes and oligodendrocytes express connexinmolecules. Connexin 43 is abundantly found in the astrocytes and ispotentially involved in various disease processes. Blood vesselendothelial cells in the optic nerve also express connexin 43.

In this study optic nerve ischaemia was induced in an ex vivo model andnerve segments placed into organotypic culture treated with connexin 43specific antisense oligodeoxynucleotides delivered in 30% F-127 Pluronicgel, or control gel.

Tissue Preparation:

Wistar rats aged 21 to 25 days postnatal (p21 to p25) were used. Thewiener rats were sacrificed by overdosing with carbon dioxide, theskulls opened in a midsagittal orientation, and the brain region caudalto the cerebellum exercised and discarded. Incisions were then madebelow the olfactory lobes to reveal the intracranial regions of theoptic nerve. Approximately 0.3 to 0.5 mm of optic nerve which spans theoptic chiasm and terminal point of the optic canal could be obtainedthrough this method. The nerves were then subjected to ischaemia asbelow.

A viable organotypic culture model of ION protocol was suggested bySundstrom et al., Drug Discovery Today 10:993-1000, (2005), working onCNS ischaemia. Prior to the experiment, 10 mL of medium prepared in afalcon tube without glucose and glutamine was bubbled with 95% N₂ and 5%CO₂ gas mixture for 30 minutes to remove all the oxygen. The dissectedoptic nerves were transferred to the oxygen glucose deprived (OGD)solution and sealed with parafilm and cellophane. The optic nerves wereincubated in ischaemic solution for two hours at 37° C. and subsequentlyreturned to organotypic culturing conditions for lengths of time asrequired.

An interphase culturing methodology used. After incubation in OGDsolutions, the optic nerves were placed onto a semi-porous membrane andinto a six well plate containing 1 ml of Neurobasal medium with B27supplement, D-glucose and L-glutamine and antibiotics (Gibco, USA). Forantisense treatment, 7 μL of Pluronic F-127 gel (#P2443, Sigma, USA)containing 10 μM AS-ODN specific for connexin 43 translational block wasadministered to cover each optic nerve. This amount was sufficient tocover the whole segment without over-flooding the tissue. For gel onlyand control groups, the same amount (7 μL) of Pluronic F-127 gel andmedium was applied to the nerves, respectively. The culture plates werethen placed into an incubator with temperature set at 37° C. with 5%CO₂. The main advantage of this culturing technique is that it ensures aconstant supply of oxygen from the top while nutrients can diffuse fromthe bottom.

After culture, the nerves were rinsed for 15 minutes in 1×PBS (# BR14,oxoid, England) and fixed in 4% paraformaldehyde (PFA) for approximatelytwo hours prior to being cryoprotected by going through 20% then 30%sucrose in PBS. The nerves were then stored in 15% sucrose in PBS untilready for further processing. To section the tissues, the optic nerveswere embedded in OCT (#4583, Tissue Tek®, USA), frozen at −20° C. andsubsequently cut into longitudinal 14 and 18 μm thick sections. Theslices were collected onto Histobond slides (#0810001, Marienfeld,Germany) and stored for further processing in a −80° C. freezer.

Swelling (oedema) was assessed by photographing optic nerves from aboveand measuring (cultured area—original area divided by original area) togive % swelling. Cell death was assessed using propidium iodide to labelthe nuclei of compromised cells. Cell death was assessed near the cutends of the nerves and in the middle region of the nerves.

FIG. 11 shows a dose response curve for antisense and control treatedoptic nerves cultured for 6 hours and 24 hours after ischemic injury.FIG. 11A shows percentage swelling, and FIG. 11B cell death assessedusing propidium iodide counts at the cut end (front) and in the middleof the nerve. Oedema is reduced in nerves with connexin 43 antisense,especially at the 10 micromolar concentration which we have previouslyshown to be optimal for crush wounds in spinal cord studies(unpublished). Cell death at both the cut end and toward the middle ofthe nerve is reduced using the antisense resulting is lower dead cellcounts in both regions in a dose dependent manner.

FIG. 12 below shows that reduction in swelling (oedema) is maintainedover time. FIG. 13 shows propidium iodide staining of dead cells in themiddle of control and connexin 43 specific AS-ODN treated optic nervesegments at 2, 6 and 24 hours after ischaemic induction. Little stainingis exhibited by the connexin 43 specific AS-ODN treated group whencompared to the controls at all three time points. The line graph inFIG. 13 shows the number of dead cells per unit area in the medialregion of the nerve for the control and AS-ODN treated optic nerves.Cell death in the control group initially increases, peaks at six hoursand then declines slightly (probably owing to tissue oedema leavingfewer cells per unit area). Only a very slight increase in cell deathafter even 24 hours in culture was noted for AS-ODN treated tissue.

Blood Vessel Segment Lengths—von Willebrand Factor Staining:

In order to demonstrate that blood vessel integrity was beingcompromised by connexin expression vessels in control and connexin 43specific antisense treated optic nerves in the ischemic model werelabelled with von Willebrand factor, an endothelial cell marker. Asvessels broke down increasing numbers of smaller segments could becounted and segment length measured. The mean length and number of bloodvessels per section was investigated in more than sixty vessels in sixseparate sections obtained from two animals for each point.

On average, the number of blood vessel segments in the controls wasfewer than that in connexin 43 specific AS-ODN treated nerves at all butthe longest time point investigated (Table 8). The bar graph (FIG. 14)shows that the mean vessel length in connexin 43 specific AS-ODN treatedoptic nerves remained relatively constant throughout the first threedays, but starting to fall by approximately 30% by Day 6. A similartemporal pattern is observed for the control group but for all timepoints average vessel segment length is significantly shorter than forthe antisense treated group.

TABLE 8 Number of Segments per Section AS Ctrl Day 1 10.8 23.5 Day 213.3 19.2 Day 3 29.7 41 Day 6 18.2 13.8

Table B shows the average number of blood vessel segments counted incontrol and treated groups. In the first three days, AS treated groups,on average, have 28˜50% fewer blood vessel segments in comparison tocontrol. Only after 6 days in organotypic culture does the AS treatednerve a greater segment count than in controls, having 24% more vesselscounted. Extended culture times may by then be having an effect.

This Example shows that prevention of connexin 43 expression followingoptic nerve ischaemia reduces oedema (reduced swelling), lesion spread(number of dead cells per unit area away from the original damage zone)and blood vessel degradation. It therefore behaves in a similar mannerto that reported in Example 1—spinal cord. It has therapeuticapplications as the same oedema and vessel loss is reported in in vivostudies (Bernstein et al., Invest Opthalmol Vis Sci 44:4153-4162,(2003)).

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe levels of skill of those skilled in the art to which the inventionpertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art, uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “consisting essentially of”, and“consisting of” may be replaced with either of the other two terms inthe specification. Also, the terms “comprising”, “including”,containing”, etc. are to be read expansively and without limitation. Themethods and processes illustratively described herein suitably may bepracticed in differing orders of steps, and that they are notnecessarily restricted to the orders of steps indicated herein or in theclaims. It is also that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Under no circumstances may thepatent be interpreted to be limited to the specific examples orembodiments or methods specifically disclosed herein. Under nocircumstances may the patent be interpreted to be limited by anystatement made by any Examiner or any other official or employee of thePatent and Trademark Office unless such statement is specifically andwithout qualification or reservation expressly adopted in a responsivewriting by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed is:
 1. A method for treating tissue edema associatedwith a vascular disorder, comprising administering a connexin mimeticpeptide comprising the amino acid sequence of SEQ ID NO:35, or SEQ IDNO:36 to a subject in an amount capable of modulating a connexinhemichannel in a tissue associated with said vascular disorder, whereintissue edema associated with said vascular disorder is ameliorated. 2.The method of claim 1 wherein said connexin mimetic peptide isadministered once.
 3. The method of claim 1 wherein the modulation ofthe hemichannel comprises inhibiting extracellular hemichannelcommunication.
 4. The method of claim 1 wherein said vascular disorderis a stroke.
 5. The method of claim 1 wherein blood vessel die back isameliorated.
 6. The method of claim 1 wherein blood vessel break down isameliorated.
 7. The method of claim 1 wherein the vascular disorder is acentral nervous system injury.
 8. The method of claim 1 wherein thevascular disorder comprises an ischemia.
 9. The method of claim 8wherein the ischemia is a tissue ischemia.
 10. The method of claim 8wherein the ischemia is a myocardial ischemia.
 11. The method of claim 8wherein the ischemia is a cerebral ischemia.
 12. The method of claim 1wherein the subject is at risk of loss of neurological function byischemia.
 13. The method of claim 1 wherein the connexin target isconnexin
 43. 14. The method of claim 1 wherein the vascular disorder issubsequent to a stroke resulting in tissue damage.
 15. The method of anyone of claims 4-6, 8-12, 1, 13 or 14, further wherein cell death ordegeneration in the central or peripheral nervous system resulting froman ischemia is ameliorated.
 16. The method of claim 1 wherein thevascular disorder is selected from the group consisting of ischemicstroke, transient ischemic attack, intracerebral hemorrhage,subarachnoid hemorrhage, thromboembolic stroke, venous thrombosis,pulmonary embolism, embolic stroke, cerebrovascular disorder, peripheralocclusive arterial disease, arteriovenous malformation, and an aneurysm.17. A method for treating tissue edema associated with a vasculardisorder, comprising administering a connexin mimetic peptide comprisingthe amino acid sequence of SEQ ID NO:35, or SEQ ID NO:36 to a subject inan amount capable of modulating a connexin hemichannel in a tissueassociated with said vascular disorder, wherein tissue edema associatedwith said vascular disorder is ameliorated, wherein the connexin mimeticpeptide is administered in connection with a vascular or coronaryprocedure performed on the subject, and further wherein the connexinmimetic peptide is administered during the vascular or coronaryprocedure.
 18. The method of claim 17 wherein the connexin mimeticpeptide is administered in connection with a heart surgery performed ona subject.
 19. A method for treating tissue edema associated with avascular disorder, comprising administering a connexin mimetic peptidecomprising the amino acid sequence of SEQ ID NO:35, or SEQ ID NO:36 to asubject in an amount capable of modulating a connexin hemichannel in atissue associated with said vascular disorder, wherein tissue edemaassociated with said vascular disorder is ameliorated, wherein theconnexin mimetic peptide is administered in connection with a vascularor coronary procedure performed on the subject, and further wherein theconnexin mimetic peptide is administered within about one hour after thevascular or coronary procedure is performed.
 20. A method for treatingtissue edema associated with a vascular disorder, comprisingadministering a connexin mimetic peptide comprising the amino acidsequence of SEQ ID NO:35, or SEQ ID NO:36 to a subject in an amountcapable of modulating a connexin hemichannel in a tissue associated withsaid vascular disorder, wherein tissue edema associated with saidvascular disorder is ameliorated, wherein the connexin mimetic peptideis administered in connection with a vascular or coronary procedureperformed on the subject, and further wherein the connexin mimeticpeptide is administered within about 2-24 hours after the vascular orcoronary procedure is performed.
 21. A method for treating tissue edemaassociated with a vascular disorder, comprising administering a connexinmimetic peptide comprising the amino acid sequence of SEQ ID NO:35, orSEQ ID NO:36 to a subject in an amount capable of modulating a connexinhemichannel in a tissue associated with said vascular disorder, whereintissue edema associated with said vascular disorder is ameliorated, andfurther wherein the connexin mimetic peptide is administered inconnection with a medical device for performing a vascular procedure.22. A method for treating tissue edema associated with a vasculardisorder, comprising administering a connexin mimetic peptide comprisingthe amino acid sequence of SEQ ID NO:35, or SEQ ID NO:36 to a subject inan amount capable of modulating a connexin hemichannel in a tissueassociated with said vascular disorder, wherein tissue edema associatedwith said vascular disorder is ameliorated, wherein the connexin mimeticpeptide is administered in combination with a second compound thatreduces tissue damage or promotes healing and further wherein the secondcompound is a growth factor or cytokine.
 23. The method of claim 22wherein the growth factor or cytokine is selected from the groupconsisting of fibroblast growth factor (FGF), nerve growth factor (NGF),neutrophin 3 (NT3), platelet-derived growth factor (PDGF), tumor growthfactor (TGF), vascular endothelial growth factor (VEGF), brain-derivedgrowth factor (BDGF), epidermal growth factor (EGF), keratinocyte growthfactor (KGF), plasmin, an integrin, an interleukin, and a semaphorin.24. A method for treating tissue edema associated with a vasculardisorder, comprising administering a connexin mimetic peptide comprisingthe amino acid sequence of SEQ ID NO:35 or SEQ ID NO:36 to a subject inan amount capable of modulating a connexin hemichannel in a tissueassociated with said vascular disorder, wherein tissue edema associatedwith said vascular disorder is ameliorated, wherein the connexin mimeticpeptide is administered systemically to the subject.
 25. A method fortreating tissue edema associated with any one of a vascular disorder, aninflammatory disorder, and a transplant or grafting procedure,comprising administering a connexin mimetic peptide comprising the aminoacid sequence of SEQ ID NO:35 or SEQ ID NO:36 to a subject in an amountcapable of modulating a connexin hemichannel in a tissue associated withany one of said vascular disorder, an inflammatory disorder, and atransplant or grafting procedure, wherein tissue edema associated withany one of said vascular disorder, an inflammatory disorder, and atransplant or grafting procedure, is ameliorated, wherein the connexinmimetic peptide is administered intravenously to the subject.
 26. Amethod for treating tissue edema associated with a vascular disorder oran inflammatory disorder comprising administering a connexin mimeticpeptide comprising the amino acid sequence of SEQ ID NO:35 or SEQ IDNO:36 to a subject in an amount capable of modulating a connexinhemichannel in a tissue associated with said vascular disorder or saidinflammatory disorder, wherein tissue edema associated with saidvascular disorder or said inflammatory disorder is ameliorated, whereinthe connexin mimetic peptide is administered intraarticularly to saidsubject.
 27. A method for treating tissue edema associated with avascular disorder or an inflammatory disorder comprising administering aconnexin mimetic peptide comprising the amino acid sequence of SEQ IDNO:35 or SEQ ID NO:36 to a subject in an amount capable of modulating aconnexin hemichannel in a tissue associated with said vascular disorderor said inflammatory disorder, wherein tissue edema associated with saidvascular disorder or said inflammatory disorder is ameliorated, whereinthe connexin mimetic peptide is administered periarticularly to saidsubject.
 28. A method for treating tissue edema associated with avascular disorder or an inflammatory disorder comprising administering aconnexin mimetic peptide comprising the amino acid sequence of SEQ IDNO:35 or SEQ ID NO:36 to a subject in an amount capable of modulating aconnexin hemichannel in a tissue associated with said vascular disorderor said inflammatory disorder, wherein tissue edema associated with saidvascular disorder or said inflammatory disorder is ameliorated, whereinthe connexin mimetic peptide is administered intraportally to saidsubject.
 29. A method for treating tissue edema associated with avascular disorder or an inflammatory disorder comprising administering aconnexin mimetic peptide comprising the amino acid sequence of SEQ IDNO:35 or SEQ ID NO:36 to a subject in an amount capable of modulating aconnexin hemichannel in a tissue associated with said vascular disorderor said inflammatory disorder, wherein tissue edema associated with saidvascular disorder or said inflammatory disorder is ameliorated, whereinthe connexin mimetic peptide is administered directly into an organ ofsaid subject.
 30. A method for treating tissue edema associated with avascular disorder or an inflammatory disorder comprising administering aconnexin mimetic peptide comprising the amino acid sequence of SEQ IDNO:35 or SEQ ID NO:36 to a subject in an amount capable of modulating aconnexin hemichannel in a tissue associated with said vascular disorderor said inflammatory disorder, wherein tissue edema associated with saidvascular disorder or said inflammatory disorder is ameliorated, whereinthe connexin mimetic peptide is administered into the cerebrospinalfluid (ICSF) of said subject.
 31. A method for treating tissue edemaassociated with a vascular disorder or an inflammatory disordercomprising administering a connexin mimetic peptide comprising the aminoacid sequence of SEQ ID NO:35 or SEQ ID NO:36 to a subject in an amountcapable of modulating a connexin hemichannel in a tissue associated withsaid vascular disorder or said inflammatory disorder, wherein tissueedema associated with said vascular disorder or said inflammatorydisorder is ameliorated, wherein the connexin mimetic peptide isadministered intracranially to said subject.
 32. A method for treatingtissue edema associated with a vascular disorder or an inflammatorydisorder comprising administering a connexin mimetic peptide comprisingthe amino acid sequence of SEQ ID NO:35 or SEQ ID NO:36 to a subject inan amount capable of modulating a connexin hemichannel in a tissueassociated with said vascular disorder or said inflammatory disorder,wherein tissue edema associated with said vascular disorder or saidinflammatory disorder is ameliorated, wherein the connexin mimeticpeptide is administered into the spinal medulla of said subject.
 33. Amethod for treating tissue edema associated with a vascular disorder oran inflammatory disorder comprising administering a connexin mimeticpeptide comprising the amino acid sequence of SEQ ID NO:35 or SEQ IDNO:36 to a subject in an amount capable of modulating a connexinhemichannel in a tissue associated with said vascular disorder or saidinflammatory disorder, wherein tissue edema associated with saidvascular disorder or said inflammatory disorder is ameliorated, whereinthe connexin mimetic peptide is administered directly into the heart ofsaid subject.
 34. A method for treating tissue edema associated with avascular disorder, comprising administering a connexin mimetic peptidecomprising the amino acid sequence of SEQ ID NO:35 or SEQ ID NO:36 to asubject in an amount capable of modulating a connexin hemichannel in atissue associated with said vascular disorder, wherein tissue edemaassociated with said vascular disorder is ameliorated, wherein theconnexin mimetic peptide is administered as a bolus dose.
 35. A methodfor treating tissue edema associated with a vascular disorder,comprising administering a connexin mimetic peptide comprising the aminoacid sequence of SEQ ID NO:35 or SEQ ID NO:36 to a subject in an amountcapable of modulating a connexin hemichannel in a tissue associated withsaid vascular disorder, wherein tissue edema associated with saidvascular disorder is ameliorated, wherein the connexin mimetic peptideis administered by sustained delivery.
 36. A method for treating tissueedema associated with a vascular disorder, comprising administering aconnexin mimetic peptide comprising the amino acid sequence of SEQ IDNO:35 or SEQ ID NO:36 to a subject in an amount capable of modulating aconnexin hemichannel in a tissue associated with said vascular disorder,wherein tissue edema associated with said vascular disorder isameliorated, and wherein the connexin mimetic peptide is administeredfor at least 72 hours.
 37. A method of any one of claim 24, or 34-36wherein the connexin mimetic peptide is administered in connection witha vascular or coronary procedure performed on the subject.
 38. Themethod of any one of claim 24, or 34-36 wherein the connexin mimeticpeptide is administered in combination with a second compound thatreduces tissue damage or promotes healing.
 39. The method of any one ofclaim 24, or 34-36 wherein the vascular disorder is associated with oneor more of coronary heart disease, coronary vascular disorder,atherosclerotic vascular disease, athersclerotic plaque rupture, and/orthromboembolic, a vascular disorder associated with hypertension,myocardial infarction, angina, ischemic heart disease, aortic disorder,peripheral arterial diseases, fibromuscular dysplasia, moyamo disease,or thromboangiitis.
 40. The method of any one of claims 24 and 34-36wherein the subject has one or more of hemostatis, thrombosis,fibrinolysis, cardiovascular disease, diabetes mellitus, an endocrinedisorder affecting the heart, cardiovascular disease associated withpregnancy, rheumatic fever, a cardiovascular disorder associated withHIV-infection, a hematological or oncological disorder associated withheart disease, a neurological disorder associated with heart disease,and a renal disorder associated with heart disease.
 41. A method fortreating a subject for tissue edema associated with an inflammatorydisorder comprising administering to said subject a therapeuticallyeffective amount of a connexin mimetic peptide comprising the amino acidsequence of SEQ ID NO:35 or SEQ ID NO:36 capable of inhibiting theexpression, formation, or activity of a connexin hemichannel, whereintissue edema associated with said inflammatory disorder is ameliorated,wherein the connexin mimetic peptide is administered systemically to thesubject.
 42. A method for treating a subject for tissue edema associatedwith a transplant or grafting procedure comprising administration tosaid subject an amount of a connexin mimetic peptide comprising theamino acid sequence of SEQ ID NO:35 or SEQ ID NO:36, capable ofinhibiting the expression, formation or activity of a connexinhemichannel, wherein tissue edema associated with said transplant orgrafting procedure is ameliorated, wherein the connexin mimetic peptideis administered systemically to the subject.
 43. A method for treating asubject for tissue edema associated with an inflammatory disordercomprising administering to said subject a therapeutically effectiveamount of a connexin mimetic peptide comprising the amino acid sequenceof SEQ ID NO:35 or SEQ ID NO:36 capable of inhibiting the expression,formation, or activity of a connexin hemichannel, wherein tissue edemaassociated with said inflammatory disorder is ameliorated, wherein theconnexin mimetic peptide is administered as a bolus dose.
 44. A methodfor treating a subject for tissue edema associated with a transplant orgrafting procedure comprising administration to said subject an amountof a connexin mimetic peptide comprising the amino acid sequence of SEQID NO:35 or SEQ ID NO:36, capable of inhibiting the expression,formation or activity of a connexin hemichannel, wherein tissue edemaassociated with said transplant or grafting procedure is ameliorated,wherein the connexin mimetic peptide is administered as a bolus dose.45. A method for treating a subject for tissue edema associated with aninflammatory disorder comprising administration to said subject anamount of a connexin mimetic peptide comprising the amino acid sequenceof SEQ ID NO:35 or SEQ ID NO:36, capable of inhibiting the expression,formation or activity of a connexin hemichannel, wherein tissue edemaassociated with said inflammatory disorder transplant or graftingprocedure is ameliorated, wherein the connexin mimetic peptide isadministered by sustained delivery.
 46. A method for treating a subjectfor tissue edema associated with a transplant or grafting procedurecomprising administration to said subject an amount of a connexinmimetic peptide comprising the amino acid sequence of SEQ ID NO:35 orSEQ ID NO:36, capable of inhibiting the expression, formation oractivity of a connexin hemichannel, wherein tissue edema associated withsaid transplant or grafting procedure is ameliorated, wherein theconnexin mimetic peptide is administered by sustained delivery.
 47. Amethod for treating a subject for tissue edema associated with aninflammatory disorder comprising administration to said subject anamount of a connexin mimetic peptide comprising the amino acid sequenceof SEQ ID NO:35 or SEQ ID NO:36, capable of inhibiting the expression,formation or activity of a connexin hemichannel, wherein tissue edemaassociated with said inflammatory disorder is ameliorated, wherein theconnexin mimetic peptide is administered for at least 72 hours.
 48. Amethod for treating a subject for tissue edema associated with atransplant or grafting procedure comprising administration to saidsubject an amount of a connexin mimetic peptide comprising the aminoacid sequence of SEQ ID NO:35 or SEQ ID NO:36, capable of inhibiting theexpression, formation or activity of a connexin hemichannel, whereintissue edema associated with said transplant or grafting procedure isameliorated, wherein the connexin mimetic peptide is administered for atleast 72 hours.
 49. The method of any one of claim 24, 34-36, 41, 42,45, or 47 wherein a hemichannel expression, formation, or activity isinhibited in endothelial cells.
 50. The method of any one of claim 24,34-36, 41, 43, 45, or 47 wherein transmission of molecules from thecytoplasm of a cell bearing the hemichannel into an extracellular spaceis inhibited.
 51. The method of any one of claim 41, 43, 45, or 47wherein the inflammatory disorder is selected from the group consistingof arthritis, rheumatoid arthritis (RA), inflammation, destruction ordamage of joints, inflammatory disorder, grave's disease, hashimoto'sdisease, rheumatoid arthritis, systemic lupus erythematosus, sjogrenssyndrome, immune thrombocytopenic purpura, multiple sclerosis,myasthenia gravis, scleroderma, psoriasis, inflammatory bowel disease,Crohn's disease, ulcerative colitis, sepsis and septic shock, andautoimmune diseases of the digestive system.
 52. The method of any oneof claim 42, 44, 46, or 48 wherein the subject is treated with atransplant or grafting procedure associated with heart failure,congenital heart disease, acquired heart disease in children, valvularheart disease, infective endocarditis, cardiomypopathy, tumors of theheart, pericardial heart disease, traumatic heart disease, pulmonaryembolism, pulmonary hypertension, cor pulmonale, and athletic heartsyndrome, peripheral arterial circulation disorder, vascular disorderaffecting an organ system, vascular disorder affecting the centralnervous system, vascular disorder affecting the brain, vascular disorderaffecting the retina, vascular disorder affecting the kidney, vasculardisorder affecting and nerves, microvascular disorder, and macrovasculardisorder.
 53. The method of any one of claim 42, 44, 46, or 48 whereinthe transplant is a heart transplant, kidney transplant, livertransplant, lung transplant, pancreatic transplant, intestinaltransplant, or a combined organ transplant.
 54. The method of any one ofclaim 42, 44, 46, or 48 wherein the transplant or grafting procedureinvolves one or more of eye tissue, skin, heart valves, bones, tendons,veins, ligaments, bone marrow transplants, dental or gum tissue,grafting or implantation associated with cosmetic surgery, grafting orimplantation associated with a hip or joint replacement procedure, andtissue grafting or implants involving stem cells.